Classifications

• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/28—Means for regulation, monitoring, measurement or control, e.g. flow regulation of redox potential
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
• • 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
  • C12Q2304/00—Chemical means of detecting microorganisms
  • C12Q2304/40—Detection of gases
  • C12Q2304/44—Oxygen
• • 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
  • C12Q2304/00—Chemical means of detecting microorganisms
  • C12Q2304/80—Electrochemical detection via electrodes in contact with culture medium

Definitions

• the present invention relates to a method for the detection of microorganisms. More particularly, the present invention relates to a simple, efficient and reliable electrochemical method for the detection of bacteria by measuring the decrease in polarographic oxygen current passing through an electroanalytical cell containing two dissimilar wire electrodes immersed in a liquid culture medium.
• the determination of whether or not a substance is contaminated with biologically active agents such as bacteria is of great importance to the medical field, the pharmaceutical industry, the public health field, the cosmetic industry, the food processing industry, and in the preparation of interplanetary space vehicles.
• One of the most widely used techniques for making this determination, especially in medical applications, has been nutrient agar plating. In this method a microorganism is allowed to grow on an agar nutrient substrate, and the growth of the microorganism is observed, at first visually and thereafter by microscopic examination. This technique, which is most commonly used clinically, requires overnight incubation of plates before results are available.
• Another technique widely used for the determination of microorganisms involves supplying a microorganism in a growth medium with carbon-14 labeled glucose or the like. See Waters U.S. Patent No. 3,676,679 and Waters U.S. Patent No. 3,935,073.
• the microorganism metabolizes the radioactive glucose and evolves C 14 O 2 , which is sampled and counted. While positive results can be obtained by this radiometric method in a relatively short period of time, this method requires the use of comparatively expensive and complex apparatus and involves the handling of radioactive materials.
• the prior art also describes a number of detection techniques based on electrochemical phenomena. Generally these techniques employ very delicate and expensive electronic equipment and are extremely difficult to use in an on-going detection program.
• One of these described methods involves the measurement of polarographic oxygen current in an electroanalytical cell.
• Cell current is a function of the dissolved oxygen content of the electrolyte, and the metabolic activity of any oxygenconsuming microorganisms present will, therefore, cause the current values to fall off.
• Hitchman Measurement of Dissolved Oxygen (1977); Fatt, Polarographic Oxygen Sensors (1976); and Norris, Methods in Microbiology (1970).
• Modern techniques of polarographic oxygen measurement rely almost exclusively on the so called Clark-type electrodes which employ a semi-permeable membrane to prevent the electrodes from contacting the solution; see Clark U.S. Patent No. 2,913,386.
• the commercially available membrane polarographic oxygen detector is presently used to determine dissolved oxygen in BOD studies, marine ecology, wastewater treatment and the like.
• the MPOD is usually constructed with an inert cathode material (gold, platinum) and a silversilver chloride reference electrode, and uses a relatively concentrated (0.3
• the electrode areas are relatively large (ca. 1.0cm 2 ) and are prevented from contacting the solution to be analyzed by a semi-permeable membrane, usually of polyethylene or polytetrafluoroethylene.
• the potential applied to the electrodes is normally about 0.8V, cathode negative. This potential must be applied to the MPOD several minutes prior to any use of the detector, and must remain applied throughout the duration of any measurements to be made.
• the MPOD is thus a "steady-state" device, in that all electrode reactions stabilize at new equilibrium values under the influence of an applied potential constant in time.
• the steady-state cell current detected under these circumstances is a measure of the dissolved oxygen content of the solution.
• the Clark-type polarograph sensors suffer from serious drawbacks which make them undesirable for the detection of microorganisms. These sensors are expensive and cumbersome to use.
• the relatively high cost of the electrodes precludes the use of a separate electrode for each sample.
• the electrode surfaces have to be sterilized between samples using a strong bactericide, and then rinsed completely with a sterile rinse solution so as not to kill organisms in or contaminate the contents of the next sample cell tested.
• Microorganisms growing in the medium use oxygen from the solution, and may possibly contribute to other redox reactions which cause the measured potential, usually greater than +100mV (cathode or platinum positive with reference to the standard calomel electrode) in sterile medium, to shift toward more negative values. Aerobic organisms are able to reduce the solution enough to yield measured potentials of -100mV to -200mV (vs. SCE). This is also the range of redox potentials where the voltammetric methods cease to function; the cell current due to dissolved oxygen is by this time very small, and is usually swamped by the residual cell current due to solution impurities and electrode imperfections. Facultative anaerobes, however, may reduce the solution extensively. An exhausted culture of P.
• mirabilis will have reduced the medium in a sealed container to a value of around -550mV (vs. SCE) before ceasing growth.
• the redox potential method by itself is not well suited to the detection of bacteria because it is relatively slow and the response will depend on the type of organism being detected.
• an electroanalytical method for detecting the presence of oxygen-consuming microorganisms in a sample comprising the steps of providing a mixture of said sample and a fluid culture medium capable of supporting microorganism growth in an electroanalytical cell equipped with two electrodes which are in contact with said mixture; applying a series of voltage pulses of substantially constant amplitude and duration across said electrodes; and measuring the resulting current prior to the trailing edge of each of said applied voltage pulses; the presence of oxygen-consuming microorganisms being indicated by a decrease in cell current which is a function of the dissolved oxygen content of said mixture.
• the present invention also contemplates a process for the detection of microorganisms as described above and additionally comprising measuring the open-circuit voltage potential across said electrodes during the interval between successive applied voltage pulses.
• Determination of the dissolved oxygen content of the cell is accomplished by pulsing the electrodes briefly with a known potential (cathode negative) and measuring the resulting current through the cell prior to the trailing edge of the applied voltage pulse.
• the growth medium in the cell is used as both the analyte and the electrolyte for the determination.
• the very low duty cycle of the pulse with respect to the overall sampling interval obviates the need for constant agitation or stirring of the sample solution required by conventional steady-state methodology, and permits the same electrodes to be used to determine the relative oxidation-reduction potential in the cell through the measurement of the open-circuit potential existing between the electrodes.
• Bacterial detection is best accomplished by measuring the decrease in pulsed voltammetric oxygen current, while information characteristic of the type of organism present is best furnished by the relative cell potential determination.
• Times-to-detection for all organisms studied vary with inoculum strength in a predictable fashion, permitting accurate quantification of the organism in question when results are compared with times-to-detection obtained using known inocula of the same organism.
• the process of the present invention provides numerous advantages compared to traditional manual methodology and present automated systems. This process requires a cell of very simple construction, provides ample opportunity for the creation of disposables, promotes automated quality control, prevents any chance of cross-contamination, and can be configured as an instrument very sophisticated in operation, yet extremely simple to operate.
• Figure 1 is a front elevation view of an electroanalytical cell useful in the process of the present invention.
• Figure 2 is a top plan view of the electroanalytical cell of Fig. 1.
• Figure 3 is a sectional view of the electroanalytical cell of Fig. 1 taken along line 3-3.
• Figure 4 is a simplified schematic diagram of an analog conditioning circuit useful in the process of the present invention.
• Figure 5 is graph showing the cell current response of a fully-grown E. coli culture at various pulse widths as a function of applied cell voltage.
• Figure 6 is a graph showing the cell current response of a sterile cell with constant applied potential as a function of elasped time since pulse application.
• Figure 7 is a graph showing the cell current response as in Figure 6 with the elapsed time extended to 300 seconds.
• Figure 8 is a graph showing the cell current response as a function of applied potential for a sterile electroanalytical cell and for a cell containing fully-grown E. coli culture.
• Figure 9 is a graph showing voltammetric cell current response for a sterile cell purged with dry nitrogen; cell current is recorded as a function of elasped time during the purge.
• Figure 10 is a graph showing the cell current response for the sterile cell of Fig. 9 purged with dry nitrogen, then purged with room air.
• Figure 11 is a schematic representation of the sampling and dilution scheme used in the preparation of electroanalytical cells and pour plates for the examples.
• Figure 12 is a graph showing normalized voltammetric cell current response as a function of incubation time for varying inoculum strengths of the organism E. coli.
• Figure 13 is a graph showing normalized cell potential response as a function of incubation time for varying inoculum strengths of the organism E. coli.
• Figure 14 is a graph showing normalized voltammetric cell current response as a function of incubation time for varying inoculum strengths of the organism E. cloacae.
• Figure 15 is a graph showing normalized cell potential response as a function of incubation time for varying inoculum strengths of the organism E. cloacae.
• Figure 16 is a graph showing normalized voltammetric cell current response as a function of incubation time for varying inoculum strengths of the organism P. mirabilis.
• Figure 17 is a graph showing normalized cell potential response as a function of incubation time for varying inoculum strengths of the organism P. mirabilis.
• Figure 18 is a graph showing normalized voltammetric cell current response as a function of incubation time for varying inoculum strengths of the organism P. aeruginosa.
• Figure 19 is a graph showing normalized cell potential response as a function of incubation time for varying inoculum strengths of the organism P. aeruginosa.
• Figure 20 is a graph showing normalized voltammetric cell current response as a function of incubation time for varying inoculum strengths of the organism S. aureus.
• Figure 21 is a graph showing normalized cell potential response as a function of incubation time for varying inoculum strengths of the organism S. aureus.
• Figure 22 is a graph showing normalized voltammetric cell current response as a function of incubation time for varying inoculum strengths of the organism S. bovis.
• Figure 23 is a graph showing normalized cell potential response as a function of incubation time for varying inoculum strengths of the organism S. bovis.
• Figure 24 is a graph showing normalized cell current response as a function of incubation time for the organism E. coli with four decades of initial inoculum concentration.
• Figure 25 is a graph showing the logarithm of the initial inoculum dilution ratio as a function of time-to-detection at a 60% detection threshold for the data shown in Fig. 24.
• Figure 26 is a graph showing normalized voltammetric cell current response as a function of incubation times for varying inoculum strengths of the organism E. coli using cells fitted with gold cathodes.
• Figure 27 is a graph showing normalized voltammetric cell current response as a function of incubation times for varying inoculum strengths of the organism P. mirabilis using cells fitted with gold cathodes.
• Figure 28 is a graph showing normalized voltammetric cell current response as a function of incubation times for varying inoculum strengths of the organism P. aeruginosa using cells fitted with gold cathodes.
• Figure 29 is a top plan view of another electroanalytical cell useful in the practice of the present invention.
• Figure 30 is a sectional view of the electroanalytical cell of Figure 29 taken along line 30-30.
• Figure 31 demonstrates the effect of a data-correcting algorithm upon raw current data from an uninoculated well.
• Figure 32 is a graph showing normalized voltammetric cell current response as a function of nculation time for the organism E. coli employing a cell having plated silver wire electrode.
• Figures 33 and 34 are graphs showing normalized voltammetric cell current response as a function if incubation time for the organisms E. cloacae and P. aeruginosa employing a cell having plated silver wire electrodes.
• the present invention detects the presence of bacteria in a suspect sample primarily by measuring the decrease in voltammetric oxygen current passing through an electroanalytical cell containing the sample and a fluid growth medium. Viable organisms capable of utilizing dissolved oxygen during metabolism will cause the detected oxygen current to decrease with continued incubation, signifying detection. Sterile inocula will evidence no such current decrease. Additional means is provided to measure the opencircuit voltage of the analytical cell in order to obtain information as to the type of bacteria (primarily aerobic or facultative anaerobic) present in the cell. Organisms that consume little or no oxygen from the growth medium, yet which have the ability to alter the solution redox potential, may be detected by noting the change in the solution redox potential with incubation, as furnished by the open-circuit cell potential measurement.
• the method of the present invention can be used to detect the presence of any aerobic or facultative organisms which consume oxygen from a liquid medium during metabolism.
• organisms include bacteria such as E. coli, E. cloacae, P. mirabilis, P. aeruginosa, S. aureus, S. bovis, K. peneumoniae, S. albus, K. oxytoca, E. aerogenes, E. agglomerans, C. freundii, P. morganii, P. stuartii, S. marcescens, Group B. Beta strep, Grp. D. Strep, and yeasts such as C. albicans.
• a small portion of a suspect sample is first introduced into an electroanalytical cell containing a liquid growth medium.
• the growth medium also serves as the primary electrolyte in the cell. Any medium which will support the growth of oxygen - consuming microorganisms may be utilized.
• Typical growth media generally contain water, a carbon source, a nitrogen source, calcium, magnesium, potassium, phosphate, sulfate, and trace amounts of other minor elements.
• the carbon source may be a carbohydrate, amino acid, mono- or dicarboxylic acid or salt thereof, polyhydroxy alcohol, hydroxy acid or other metabolizable carbon compound.
• the carbon source will comprise at least one sugar such as glucose, sucrose, fructose, xylose, maltose, lactose etc.
• Amino acids such as lysine, glycine, alanine, tryrosine, threonine, histidine, leucine, etc. also frequently comprise part of the culture media carbon source.
• the nitrogen source may be nitrate, nitrite, ammonia, urea or any other assimilable organic or inorganic nitrogen source.
• An amino acid might serve as both a carbon and a nitrogen source. Sufficient nitrogen should be present to facilitate cell growth.
• a variety of calcium, potassium and magnesium salts may be employed in the growth medium including chlorides, sulfates, phosphates and the like.
• phosphate and sulfate ions can be supplied as a variety of salts.
• materials are conventional in fermentation media, the selection of specific materials as well as their proportions is within the skill of the art.
• minor elements which are present in trace amounts are commonly understood to include manganese, iron, zinc, cobalt and possibly others. Due to the fact that most biologically active species cannot function in strongly acidic or strongly alkaline media, suitable buffers such as potassium or ammonium phosphates may be employed, if desired, to maintain the pH of the growth medium near neutrality.
• Examples of well known growth media which may be used in the present invention are peptone broth, tryptic soy broth, nutrient broth or thioglycolate broth.
• Tryptic soy broth-based medium (6B Medium, Johnston Laboratories Inc., Cockeysville, Md.) has been found to work well.
• the amount of growth medium provided in the electroanalytical cell is not overly critical. 5.0cc of 6B medium has proven very effective.
• the analytical cell useful in the process of the present invention may be of any conveniently size and shape.
• the cell can be formed from any materials normally used in the manufacture of electroanalytical cells such as glass, plastic and the like. Any material which does not affect the growth the microorganisms or the measurement of electrochemical phenomenon in the cell can be employed.
• the electronalytieal cell useful in the process of the present invention comprise a plastic container of the general configuration shown in Figs. 1-3. Cell volume may vary according to the cell design and is not critical. A cell of the type shown in Figs. 1-3 has been effectively used at a capacity of about 10-15 mis. In the preferred manner of operation a number of these cells can be utilized in the form of an array to permit testing of multiple samples.
• the electroanalytical cell is also equipped with two electrodes in electrical contact with the growth medium.
• the working electrode is normally a noble metal, for example, gold, silver or platinum. When only voltametric measurements are to be taken gold, platium or silver are preferred for the cathode. When potentiometric (redox) measurements are also taken gold should not be used as the cathode material.
• the reference electrode is preferably pure silver (99.95% or better) electr ⁇ lyzed in place using a basic electrolyte to deposit Ag 2 O on the silver. Silver chloride may be electrolytically deposited from HCl solution to form the alternative Ag/AgCl reference electrode, but this electrode has been found less stable in this application.
• the electrodes may be used in any convenient form. Preferred are wires of the above materials although other forms such as printed circuit traces can be used. Most preferred are U-shaped staples inserted through the bottom of the cell as best seen in Fig. 3.
• the electrodes can be of other conventional forms including spaced apart vertically disposed hairpin shaped electrodes.
• the electrode wire diameter is not critical. Wires as small as 0.010" may be used, provided their frangibility and low sensitivity can be tolerated. Wires approaching 0.040" probably represent a practical upper limit since these materials are quite expensive. Preferred electrode diameters are in the range of from about .015 to .050", with about 0.020" to 0.040" being most preferred. Electrode lengths are likewise noncritical.
• lengths of from about 0.5cm to 2.0cm and preferably about 1.0 to 1.5 cm are suitable.
• the wires may be separated by about 0.5 to 2.5cm and preferably about 1.0 to 2.0 cm. It will be apparent to those skilled in the art that solid precious metal wires can be replaced with less expensive wire electroplated with the precious metals of choice.
• the electrode pair may be covered with a porous gel, preferably a nutrient gel such as tryptic soy agar (TSA).
• a porous gel preferably a nutrient gel such as tryptic soy agar (TSA).
• Other gel materials which may be used include gelatin, dextran gel, carrageenan gel and the like. Best results are obtained when the gel just covers the electrodes.
• the main benefit of the gel is to reduce measurement baseline drift sometimes caused by the introduction of biological samples (urine, etc.), presumably by preventing the migration of large charged molecules to the electrodes.
• the quantity of gel is not critical; 1.0cc of TSA has served to just cover the electrodes in the type of cell shown in Figs. 1-3. It may be appreciated that some ionic conduction is necessary in the gel; hence its equivalent conductance when saturated with growth medium/electrolyte should approach that of the medium alone.
• the electrode pair may also be isolated from the effects of large charged molecules by positioning a layer of porous material such as ordinary filter paper over the electrodes. While this will not prevent contact of the electrodes with the analyte or even with the microorganisms in the sample, it will limit migration of large charged molecules to the electrode region.
• the reference electrode can be isolated from the analyte mixture by the use of a salt bridge or other conventional means. In this manner it is possible to employ a single reference electrode in conjunction with a plurality of working electrodes in separate analytical half cells.
• a series of voltage pulses of substantially constant amplitude and duration are applied across the electrodes.
• the voltage amplitude of the pulses can vary from about -0.35 v. to about -0.90v. Preferred is an applied voltage pulse of about -0.70v.
• the pulse duration should be at least about 600 milliseconds.
• the upper limit of the pulse duration is not critical. As a practical matter, times much over about 3 seconds result in a reduced current signal but may be used.
• the pulse duration can be from about 800 to 2000ms. with about 1200 ms. being most preferred.
• the pulse interval is not critical and should be short enough to follow the biological changes long enough to allow the cell to approach equilibrium conditions for redox potential measurements in the pulse intervals. Times of from about 5 min. to 20 min. are suitable. A pulse interval of about 10 minutes is preferred.
• the analytical cell and its contents should be held at a constant temperature, preferably 37° C ⁇ 0.2° C. It is understood, however, that not all biologically active agents exhibit maximum growth within the cited temperature range. If it is of interest to determine whether or not a specific microorganism which grows better at some other temperature is present, then the temperature at which the organism in question exhibits maximum growth should be employed.
• FIGs. 1-3 The construction of a typical analytical cell useful in the process of the present invention is shown in Figs. 1-3.
• a semi-flexible plastic container (1) receives the two wire electrodes (2,3) in the form of U-shaped "staples" inserted through the bottom of the container.
• the working electrode (2) is analytical-grade platinum, 0.035" diameter.
• Reference electrode (3) is 0.040" diameter Ag/AgO.
• the electrode pair is covered with a nutrient gel (4) such as tryptic soy agar (TSA).
• TSA tryptic soy agar
• (5) is also present in the cell to serve as the primary electrolyte and to facilitate the growth of microorganisms.
• a data-gathering cycle begins with the selection of this particular cell. After a short delay to allow the cell selection relays to settle, a potential reading is taken with relay 11 open via operational amplifiers OA1 and OA2. Because the cell redox potentials on platinum are bipolar with respect to the reference electrode (+150mv to -550mv) with incubation of a facultative anaerobe, provision is made to offset the potential reading using OA2 to present a unipolar, positive signal of adjustable gain to the computer A/D converter.
• relay 11 closes, and a positivegoing pulse derived from the computer D/A converter is applied to OA3.
• the inverted, unity-gain output of OA3 causes current to flow through the selected cell in proportion to the dissolved oxygen content.
• the cell current is sensed by OA4, connected as a current-to-voltage converter.
• the output of OA4 is sampled by a second input of the A/D converter card prior to the termination of the voltage pulse from OA3. All relays are switched off and allowed to settle prior to the selection of the next cell to be tested, at which point the process begins again for the newly selected cell. After all cells have been tested, all relays are deselected while the programmed time interval between readings, usually ten minutes, is allowed to elapse.
• the voltage signal input resistor (R inp , OA1 in Figure 4) has the value of 2.2Megohms. Although this is by no means an electrometric input resistance as is normally employed to measure electroanalytical cell potentials, it must be remembered that the cell is loaded with this resistance for only a few hundred milliseconds before the voltage reading is stored, and that the following voltage pulse drives the cell far from equilibrium in any case.
• the 2.2M resistor provides a good compromise between cell loading and noise pickup in the cell environment.
• the cell current and potential values measured respectively during and between the successive applied pulses can be compared to the initial values to determine when the threshold of detection has been reached. This process is facilitated by normalization of the collected values as described more fully in Example 2. When the current level has fallen to a predetermined percentage of the initial value, e.g., 60-80%, detection is found to have occurred. It is also possible to make determinations by comparing the collected data to that obtained in a separate reference well.
• the method of the present invention can be used in any application where the detection of microorganisms in a sample is desired. This method finds particular utility in the detection of bacteria in biological fluids such as blood, urine and the like.
• bacterial testing can include screening, identification, and antibiotic susceptibility testing.
• Other areas of utility include the detection of microorganism contamination in food, pharmaceutical and cosmetic products.
• This example demonstrates the selection of optimum values of the voltage pulse amplitude and duration.
• a single sample cell of an array consisting of 8 cells as shown in Figure 1 was prepared using 1.0cc of molten TSA (90° C) transferred to the cell via sterile syringe. 5.0cc of a fully-grown E. coli culture in 6B medium (oxygen depleted) was similarly transferred to the cell after the agar had solidified.
• the cell array was placed in a warm-air incubator at 37° C and allowed to equilibrate for 40 minutes, at which time readings were begun under computer control.
• a voltage scan of the cell was obtained and plotted for each chosen pulse width. The interval between pulses was approximately 10 seconds, depending somewhat upon the selected pulse width.
• Figure 8 illustrates a voltage scan overlay for two cells, one containing 1.0cc of a fully-grown culture of E. coli in 6B, the other containing sterile 6B medium, both incubated at 37° C for 4 hours prior to measurement.
• a pulse width of about 1300ms was employed.
• the cell responses are observed to separate at about -0.35V, reach maximum divergence near -0.75V, and converge again about -0.90V.
• the detection of microorganisms then, requires a pulse potential near -0.75V for best efficiency under these conditions.
• a single cell of the 8-cell array was prepared as usual, containing 1.0cc TSA and 10.0cc 6B medium. The cell was incubated at 37° C for 30 minutes, then read every 60 seconds for 10 minutes. Dry nitrogen gas (Matheson, High-Purity) was then bubbled through the cell by means of a disposable glass capillary pipette. Readings were continued until the cell current dropped to a reasonably constant value. In a separate experiment conducted in the same cell, nitrogen was again bubbled through the cell, and readings begun just prior to reaching the purge plateau.
• electrochemical measurements were carried out in an 8-cell array using platinum (0.035”) and silver/silver oxide (0.040") wire electrodes of 1.0cm length separated by 1.0cm. Prior to each run, the cells were carefully rinsed with deionized water and vigorously shaken to dislodge the larger droplets. 1.0cc TSA (Trypic Soy Agar, 40.0g/l, BBL, Cockeysville, MD) at about 90° C was then transferred to each cell using a sterile 3.0cc syringe with 18ga. needle. The array was then covered, and the agar allowed to solidify.
• TSA Transpic Soy Agar, 40.0g/l, BBL, Cockeysville, MD
• 5.0cc sterile 6B medium Johnston Laboratories, Inc., Cockeysville, MD
• 0.1cc of a 4.5g/20ml sterile glucose solution and/or 0.1cc of a 1.5g/20ml sterile glycine solution as desired.
• the prepared cells were covered and set aside while the inoculum dilutions and pour plate dilutions were prepared.
• Fresh cultures of the organisms to be studied were prepared in 6B medium the day before each test, and allowed to incubate at room temperature until needed.
• Previously prepared sterile 20cc vials fitted with rubber septa and aluminum closures containing 9.0cc TSB (27.5g/l,BBL) were used for all inoculum dilutions.
• Similar 100cc vials containing 99.9cc 1/2-strength TSB were used to prepare pour plate dilutions.
• the test was initiated by preparing a sterile 1.0cc syringe containing about 0.5cc of the overnight culture of the desired organism. This culture was added dropwise to a 9.0cc dilution vial with agitation until visual turbidity was achieved.
• 1.0cc was removed from this vial and used to inoculate a second (X 0.1) vial containing 9.0cc, achieving 1:10 dilution.
• 1.0cc from this vial was used to inoculate a third (X 0.01) vial, which was in turn used to inoculate a fourth (X 0.001).
• 0.1cc was removed from the X 1.0 vial and used to inoculate one 99.9cc vial to obtain 1:1000 dilution for pour plate preparation.
• 0.1cc was withdrawn from the X 0.01 vial and used to inoculate a second 99.9cc vial to obtain 1:100,000 dilution.
• a fifth vial containing 9.0cc TSB was used as the source of sterile, control inocula.
• 1.0cc of the X 1.0 vial was then transferred to cell (1) of the array.
• Cells (2) and (3) received 1.0cc each from the X 0.1 dilution vial; cells (4) and (5) were inoculated with 1.0cc from the X 0.01 vial, while cells (6) and (7) each received an inoculum from the X 0.001 vial.
• Cell (8) was inoculated with 1.0cc sterile TSB to serve as the control.
• the inoculated cells were placed in a warm-air incubator held at 37° C ⁇ 0.2, connected to the analog conditioning electronics, and the measurements begun. No preinoculation incubation of the array was employed. Cell current and cell potential readings were obtained at 10-minute intervals using a pulse amplitude of -0.70V and a pulse duration of 1200 milliseconds.
• Duplicate pour plates were prepared containing 10-15ml TSA (40g/l) using 1.0cc and 0.1cc from each of the 99.9cc dilution vials to yield pairs of plates at 1:10 3 , 1:10 4 , 1:10 5 , and 1:10 6 dilutions. The plates were allowed to harden at room temperature prior to 24-hour incubation. Details of the sampling and dilution scheme are set out in Figure 11.
• the same normalization time (T1) is used for all samples.
• the normalized current values X(T,N), now ranging nominally from 0 to 100, are then scaled for plotting through division by a scale factor, herein 2.0, so that all data together with the machine-generated coordinate time axis will fit on the 80-character CRT/printer line. Cell currents for each sample are thus easily presented as a percentage of the normalized current value, usually taken as the current value observed after 30 minutes experiment time.
• the translated values Z(T,N) are then scaled to page width by dividing by the scale factor, herein 3.5, and then printed.
• Potential normalization is usually performed at 60-100 minutes after the experiment has begun. Cell potential readings require about 60 minutes longer, on average, to stabilize than do the current readings.
• detection of the organism is said to occur when the pulse voltammetric cell current has fallen to 80% of its value at the normalization time interval. Potential measurements are considered positive when a relative normalized value of 20 is attained. AH values obtained for cell (1) were used as high-inoculum markers only, and do not appear in the results.
• This example demonstrates the detection and quantification of E. coli.
• a fresh overnight culture of E. coli in 6B medium was used as the inoculum source.
• the sampling and dilution scheme of Figure 11 was employed to prepare sample cells and pour plates,
• the sample cell medium was enriched with 0.1cc (of the) 4.5g/20ml glucose stock solution. Incubation of all vials, sample cells and plates was at 37° C. Cell readings were continued for 8 hours.
• Pulsed voltammetric current responses for three decade dilutions of the organism plus control are shown in Figure 12.
• the related cell potential results are presented in Figure 13.
• the Y-axis indicates potential increasing in the negative direction.
• the short plateau evidenced at relative potential values of 30-40 probably indicates a change in the metabolism of the organism triggered by the reduced oxygen tension in the medium.
• Both cell parameters give times-to-detection which vary in a predictable manner with inoculum strength.
• Pour plate results indicate that 1.0 x 10 cfu/ml E. coli were present in the freshly inoculated X 0.1 cell. Times-to-detection for the duplicate cell current and potential measurements are presented in Table 1.
• This example demonstrates the detection and quantification of E. cloacae.
• a fresh culture of E. cloacae was incubated overnight at room temperature to serve as the inoculum source.
• the sampling and dilution scheme presented in Figure 11 was used to prepare duplicate sample cells and pour plates.
• the cell medium was enriched with 0.1cc glucose stock solution. All incubations were performed at 37° C.
• the inoculated cell array was covered with clean aluminum foil, placed in the incubator, and connected to the analog conditioning electronics moments before the start of the test. The test was continued for 520 minutes (8-2/3 hours).
• This example demonstrates the detection and quantification of P. mirabilis.
• a fresh overnight culture of P. mirabilis in 6B medium was used as the inoculum source.
• Sample cells and pour plates were prepared as per the sampling and dilution scheme presented in Figure 11.
• 5.0cc 6B medium was enriched with 0.1cc sterile glucose stock solution in each of the cells. All incubations were performed at 37° C. The electroanalytical measurement was begun immediately following cell inoculations without preinoculation incubation, and was continued for 540 minutes (9 hrs).
• Pulsed voltammetric cell current responses for the three decade dilutions of the organism are presented in Figure. 16.
• Cell responses appear normal as oxygen is consumed and cell current falls to the residual level, then rapid vertical transitions appear lasting only 20-30 minutes. Cell current values seem to stabilize following these events, but do not return to pre-transition levels.
• Cell potential responses are shown in Figure 17. Again, a slight plateau is observed at normalized relative potentials between 30 and 40 units. Instead of the rapidly increasing negative potentials noted for E. coli and E. cloacae, P. mirabilis potentials fall sharply after a slight increase following the plateau. The time intervals noted for this potential decrease correlate well with the observed vertical transitions in cell current previously noted. Since P.
• the silver/silver oxide electrode is noticeably blackened by exposure to P. mirabilis for extended periods.
• the electrodes do not seem to be permanently damaged by such exposure, and may be returned to their initial condition by careful washing and wiping of the electrode surfaces.
• the discoloration can be removed only by mechanical poEshing.
• the X0.1 cells (2) and (3) contained 2.0 ⁇ 10 cfu/ml of P. mirabilis at inoculation as determined by duplicate pour plate counts. Times-to-detection for cell current and cell potential are listed in Table 3.
• This example demonstrates the detection and quantification of P. aeruginosa.
• a freshly inoculated vial of 6B medium was incubated overnight at room temperature for use as the source of inocula.
• the sampling and dilution scheme illustrated in Figure 11 was employed to prepare sample cells and pour plates.
• the sample cell medium was enriched with 0.1cc sterile 1.5g/20ml glycine stock solution in each cell. All cells and plates were incubated at 37° C. Electroanalytical cell readings were begun immediately after inoculation and were continued for 490 minutes (8 1/6 hours).
• Cell current response of the organism with decade dilution and of the control cell containing sterile medium are shown in Figure 18. Normal cell current behavior is observed.
• the related cell potential responses are presented in Figure 19. Because P. aeruginosa is a relatively slow growing obligate aerobe, cell potential response at each inoculum level changes more slowly and reaches a limiting value of considerably less amplitude than do the facultative anaerobes. Pour plate counts in duplicate were used to determine the initial inoculum level in the X0.1 cells as 7.7 ⁇ 10 4 cfu/ml.
• This example demonstrates the detection and quantification of S. aureus.
• a freshly inoculated vial of 6B medium was incubated overnight at room temperature to serve as the source of all inocula.
• the sampling and dilution scheme of Figure 11 was again employed to prepare sample cells and pour plates used in the test.
• the growth medium in each cell was enriched with 0.1cc sterile glucose stock solution. All incubations were carried out at 37° C in a warm-air incubator. cell current and potential readings were recorded every 10 minutes under computer control. The experiment was continued for 480 minutes (8 hours).
• cell current response for the organism at decade inoculum levels is shown in Figure 20. Normal cell current behavior is obtained.
• the related cell potential variations are presented in Figure 21. Note that very little potential change occurs with continued growth of S. aureus; threshold detection is barely achieved.
• Figure 22 Normal current response is observed prior to the current minimum at each inoculum level. Values recorded after each minimum rise more rapidly then usual. cell potential responses are shown in Figure 23. S. bovis causes little change in the potential observed as growth progresses, save for the small singularity usually observed in conjunction with the current minima in Figure 22. Duplicate 24-hour pour plate counts indicated 6.5 ⁇ 10 5 cfu/ml to be present in the X0.1 cells initially. Times-to-detection for the detection methods are presented in Table 6.
• This example demonstrates the extended quantification of E. coli using pulsed voltammetric detection.
• a fresh overnight culture of E. coli in 6B medium was used as the inoculum source.
• Dilution vials and pour plates were prepared with reference to Figure 11, except that dilution vials were prepared out to a dilution ratio of 1:10 6 .
• the growth medium in each of the cells was enriched with 0.1cc sterile glucose stock solution.
• the first seven cells in the 8-cell array each received 1.0cc from the appropriate dilution vial as inoculum.
• cell (8) received 1.0cc sterile TSB inoculum as the control.
• cell (1) was used as a high-inoculum marker only, since the cell assembly requires at least 30 minutes to attain temperature equilibrium.
• cell (2) results were anomalous with respect to detection time, probably due to slight contamination of the cell walls or electrode surfaces during reconditioning, and are not shown.
• time-to-detection is seen to vary linearly with the logarithm of inoculum strength. Note that the detection threshold has been reduced to 60% of the value observed at the time of normalization; this provides more dependable time-to-detection values in prolonged tests where the slight downward baseline drift with time can generate detection times slightly shorter than the correct values.
• Pulsed voltammetric detection of the test organisms compares well with detection based upon the BACTEC system; E. coli and E. cloacae are detected with approximately equal facility by both methods. P. mirabilis and most notably, P. aeruginosa are. detected significantly faster using the cell current measurement. Detection of S. aureus by the cell current method is about 40 minutes faster then BACTEC, while S. bovis detection is accomplished about 1 hour sooner by the BACTEC system. cell potential detection of organism growth compared to either the cell current determination or to the BACTEC system leaves much to be desired. Results can be quite unreliable for organisms such as S. aureus (Fig. 21) and S. bovis (Fig.
• E. coli 1.0 ⁇ 10 5 cfu/ml 120 120 120 160 180 1.0 ⁇ 10 4 cfu/ml 180 150 160 220 230 1.0 ⁇ 10 3 cfu/ml 240 200 200 260 270
• the cells were prepared with either 1.5.cc or 2.0cc TSA and 5.0cc enriched 6B medium, and were tested with various organisms under the same conditions as for the previously noted experiments. No electrode pretreatment was employed. The pulse amplitude (-0.70V) and the pulse duration (1200ms) were the same as in the previous examples. Duplicate pour plates were also prepared for these experiments. cell current data normalization was carried out as previously described. Fresh overnight cultures of E. coli, P. mirabilis, and P. aeruginosa in 6B medium were used as inoculum sources. All sample and pour plate preparations were preformed with reference to Figure 11. cell current readings were obtained at 10-minute intervals. No pre-inoeulation incubation was employed.
• This example demonstrates a clinical trial of the pulsed voltammetric detection technique of the present inventor for the detection of significant bacteriuria.
• the test was conducted in conjunction with Sinai Hospital of Baltimore. Over the 33-day period of the study, 389 urine samples were collected from Yale and tested at Johnston Laboratories using the pulsed voltammetric technique in parallel with the BACTEC radiometric system. TSA pour plates were employed to cheek actual organism counts.
• the test was carried out as follows. Urine specimens sampled following Park collection and planting were picked up from the hospital at approximately 11:00AM each day. Prior to sample arrival at JLI, the 16-cell assembly to be used with the Pulsed Voltammetric instrumentation was filled with scalding hot water and allowed to stand for at least ten minutes. The assembly was then rinsed twice with sterile deionized water, then shaken vigorously to dislodge any large water droplets. 1.0cc sterile Tryptic Soy Agar (40.0g/l; BBL or DIFCO) at about 95° C was then added by sterile syringe to each cavity of the assembly.
• the assembly was then covered with a double thickness of aluminum foil sterilized with isopropanol, and the agar allowed to solidify.
• 5.0cc of sterile TSB (27.5g/l; BBL or DIFCO) containing Dextrose (2.5g/l; MALLINCKRODT or J.T. BAKER) was then added to each cavity via syringe, and the cover replaced.
• urine samples were cataloged as to JLI daily and consecutive sequence numbers, Yale reference number, and gross physical characteristics.
• 1.0cc of each urine specimen was inoculated via syringe into one cell of the assembly.
• 1.0cc was used to inoculate a septum-fitted vial of nominal 50cc capacity containing 5.0cc of JLI 4A Urine Screening Medium (JLI B/N 037901U-1.5uCi/vial) for use in the parallel BACTEC study.
• 0.1cc of each specimen was inoculated into previously refrigerated, septum-fitted vials containing 99.9cc 1/2-strength TSB to obtain
• the inoculated cell assembly was placed in a 37° C warm air incubator without agitation and pulsed voltammetric testing begun under computer control. Test values were recorded for all samples at 10-minute intervals. Data normalization was based upon sample data values recorded after the first 10 minutes for tabulation; all data values recorded for each sample were ultimately expressed as a percentage of the 10-minute value. A sample was considered to be positive when the normalized data value for that sample at any given time interval after normalization fell below 70 or rose above 140. The latter criterion was employed to permit detection of some highly positive (ca. 10 cfu/ml) samples which produced data minima slightly greater than 70, yet which interfered with normal operation sufficiently to produce data maxima over 140.
• the pulsed voltammetric method detected 80 (95.24%). Of the four samples missed by the pulsed voltammetric technique, two were missed by BACTEC as well. One of these is known to be from a patient receiving antibiotic therapy; the other contained S. Aureus. The remaining two false negative samples contained P. Aeruginosa and an unspecified yeast. The BACTEC system detected 76 (90.48%) of the 84 samples considered elineally significant.
• the pulsed voltammetric detection technique properly identified 95.24% of urines considered significant in the study, yielding a total sample false negative rate of 1.27%, with a false positive rate of 4.78%.
• the BACTEC system detected 90.48% of significant urines, with a total sample false negative rate of 2.55% and a 1.27%, with a false positive rate of 4.78%.
• the BACTEC system detected 90.48% of significant urines, with a total sample false negative rate of 2.55% and a false positive rate of 1.27%.
• BACTEC required 3 hours to achieve the reported level of performance; the pulsed voltammetric technique required 4 hours.
• FIG. 29 and 30 Another cell assembly was constructed to permit the use of disposable plated wire electrodes.
• a single cell of the 16-cell assembly is presented in Figures 29 and 30.
• Oxygen-free hard copper wire plated with 50 microinches silver was used as the electrode material.
• the identical electrodes (6) pass through the Plexiglas (TM) lid (7) and are supported by a polycarbonate wire support (8).
• Contact with each electrode is made by capturing the bent upper end of each electrode wire between a bottom beryllium copper plate (9) and a like upper contact (10).
• the contact assembly is held in place by screws (11) and Nylon spacers (12). Connection to the electronics is by means of flexible insulated wires (13) soldered to the upper contacts. All working parts of the electrode assembly are thus attached to tray top (7).
• the top is fabricated to fit a 16-well plastic tray (14) which contains the growth medium and inoculum.
• the contact assemblies are actually disposed as shown in the top view, arranged as two rows of eight sets each.
• the electrode wires (6) are bent in "hairpin" fashion so as to permit the ends of the wires to be clear of the solution (15). This is to prevent any spurious responses due to the high electric field which could be generated at a sharp wire end, and also to insure that the copper base metal does not come into contact with the solution at the exposed end.
• This cell assembly was employed for Examples 12-14.
• Silver plated copper wire was supplied hard-drawn by Sigmund Cohn, NY, on spools.
• the spooled wire was first straightened by reverse-bending and tensioning, and then cut into pieces about 2.5 inches in length. These were bent in an "L" shape to accommodate the tray lid and contact assembly, and were ultrasonically cleaned in methanol for 15 minutes.
• the wires were then air dried between Kimwipes in a fume hood. Rubber gloves were worn to transfer the dried wires to clean, dry polypropylene bottles for storage. Sterile rubber gloves were also worn when assembling the wires into the tray top and contact assemblies.
• the bottom "hairpin" bend was made last, with the wire held in the contact assembly and aligned by the wire support. Finally, excess wire was trimmed from the exposed end so as to leave about 1/4" clear of the solution.
• the correction algorithm employed adjusts the raw data prior to the normalization process described earlier.
• the purpose of the algorithm is to "add back" an inverse exponential to compensate for the observed decline in electrode current with time.
• the algorithm has the functional form:
• a fresh overnight culture of E. coli in 6B medium was used as the inoculum source.
• the sampling and dilution scheme presented in Figure 11 was employed to prepare sample cells and pour plates.
• TSB with dextrose (30.0g/l) was used as the medium for detection as described above.
• AE incubations were performed at 37 degrees C. cell readings were continued for 6 hours.
• a fresh overnight culture of E. cloacae in 6B medium was used as the source for all inocula.
• the sampling and dilution scheme presented in Figure 11 was used to prepare sample cells and pour plates. Tryptic soy broth with dextrose (30.0g/l) was used as the growth medium for detection. All incubations were performed at 37 degrees C. cell readings were continued for 7 hours.
• the technique of the present invention is thus shown to provide competent detection of significant bacteriuria, with clinically acceptable levels of false negative and false positive results.
• the rapidity and sensitivity of the method compare favorably with parallel results obtained using the BACTEC system.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Genetics & Genomics (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• Sustainable Development (AREA)
• Biomedical Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• Physics & Mathematics (AREA)
• Toxicology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=21960480

Family Applications (1)

Country Status (7)

Cited By (7)

Citations (15)

• 1980
  • 1980-06-18 CA CA000354285A patent/CA1158720A/en not_active Expired
  • 1980-06-18 BE BE0/201079A patent/BE883881A/fr not_active IP Right Cessation
  • 1980-06-18 MX MX182829A patent/MX153017A/es unknown
  • 1980-06-18 IT IT22869/80A patent/IT1141002B/it active
  • 1980-06-18 NZ NZ194089A patent/NZ194089A/en unknown
  • 1980-06-18 WO PCT/US1980/000755 patent/WO1980002849A1/en unknown
  • 1980-12-30 EP EP80901311A patent/EP0030962A1/en not_active Withdrawn

Patent Citations (16)

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events