DE1919271A1 - Apparatus and method for measuring gases - Google Patents

Description

Apparatus and method for measuring gases

The conventional methods of plaguing bacteria with The help of bacterial cultures is time consuming (they require a minimum of 24 hours) and costly (because of the Work performance) and are not as accurate as one would like could wish.

Studies on the activity of the catalase enzyme have been carried out in the past. In such investigations, the bacteria and hydrogen peroxide were brought together, and a reaction between the catalase present in the bacteria and the hydrogen peroxide was triggered, which caused the release of oxygen. The released oxygen entered the free volume above the hydrogen peroxide solution and caused an increase in the gas pressure. Attempts have been made to measure this change in gas pressure (with a manometer); However, the response of this method has clear limits that prevent the dynamic measurement of the reaction between catalase and hydrogen peroxide _s very fast runs. " The inherent lack of sensitivity of this type of measurement- speaks against its use. for the qü

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tive
/ Measurement of catalase activity. '

The catalase activity has also been measured quantitatively by titrating the remaining hydrogen peroxide a preselected reaction time »However, this method is cumbersome and inconvenient, and would be very difficult to use for dynamic measurement. There are none Possibility to continuously record the reaction.

There is a constant and urgent need for a sensitive, quantitative probe for bacteria faster response speed for use in countering biological warfare, in the investigation Water pollution, In food processing, in the identification of pathological conditions and for the regulation of environmental conditions. There is also a Lack of means for dynamic and continuous observation and recording the reaction between catalase and peroxide. These requirements are met by the various designs of the present invention met.

Most of the pathogenic bacteria that are transmitted through liquids or the atmosphere are aerobic or facultative anaerobic bacteria which, with very few exceptions, contain the enzyme catalase. Likewise, catalase is found in most animal cells, such as skin cells, erythrocytes, liver cells, and kidney cells. Therefore, urine and other body fluids may contain catalase when cells, bacteria or products of destruction of cells containing catalase are present. Hence, the presence of significant amounts of catalase would indicate that some unusual destruction of catalase-containing cells is occurring or has occurred in the body, or that significant amounts of bacteria are present. It is already known that this enzyme is extremely poor in its ability to disproportionate of hydrogen peroxide. The catalyzed bacteria are released , for example

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with about 1 to 2 % of the total protein content, which is equivalent to an amount of about 10 ^ to 10 catalase molecules per orpanism.

It has been found that a polarographic membrane cell (an anode and a cathode, which are electrically connected to each other via a suitable electrolyte, where the cathode is adjacent to a thin outer wall of a material, which is permeable to oxygen) can be used successfully can get an increase in a hydrogen peroxide solution

lh 3 /

of even less than 10 molecules per cm of oxygen / quantitative to be determined when using the cathode configuration according to the present invention. This increase in that Partial pressure of oxygen would result from the disproportionation of a hydrogen peroxide solution as a result of its presence of catalase molecules. It has been determined that the accuracy of this measurement is not due to the presence the hydrogen peroxide solution (or a hydrogen peroxide generating solution) can be affected as long as the permeable membrane, which forms part of the outer wall of the cell, is impermeable to hydrogen peroxide, is not porous and has a permeability to oxygen equal to or greater than icT cm / eec.

The presence of catalase from bacteria or various animal cells cause the hydrogen peroxide to decompose very quickly into oxygen and water. The partial pressure of oxygen the hydrogen peroxide solution itself quickly increases to a value which is the amount and the integrated Activity of the catalase enzyme present is proportional. in the If bacteria are found, it is assumed that this increase in the partial pressure of oxygen increases the number of those present Bacteria in the hydrogen peroxide solution is proportional when it is known that no other source of catalase is to be considered must be pulled. It has been found that the passage of oxygen through a selectively permeable membrane which is in contact with a hydrogen peroxide solution

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Oxygen partial pressure in the hydrogen peroxide solution is linearly proportional to ν. Through the oxygen-permeable membrane * \ ^! Oxygen gas entering a polarographic cell is generated. a cell current that can be measured and recorded. *.

As part of this invention, devices have been created *; in order to set a reproducible small volume (ie less than 0 *} - ^ cm ^) which can be placed adjacent to the selectively permeable membrane which covers the oxygen electrode j (cathode) of the polarographic cell. In this way, the presence of even a small number of ^,? Bacteria in a hydrogen peroxide solution produce the partial pressure of oxygen effectively in this selected volume, which is isolated from the "oxygen-sensitive cathode" and can grow to values of more than a few atmospheres of the oxygen partial pressure, and such supersaturation with oxygen can be sustained for long periods of time be ten if the volume is defined by a gas-tight walls in the cases where the reproducible small volume is not defined by a gas-tight walls, the oxygen can. '■ [j partial pressure usually does not exceed one atmosphere, without:''that oxygen escapes from the measuring range j

Although it is preferable that the measurement range be a closed -. Volume, it is possible to carry out the process of the invention with the hydrogen peroxide solution to the atmosphere; is open. Likewise, it is not necessary to apply the above _ \ small reaction volume, when a large number to be determined by bacteria, such as 1st it reindeer at a urinä- _E infection case. ■■ "- .. >

The invention is illustrated by the following description together with the attached illustrations.

Fig. 1 is a schematic representation of a system for? ■ '"""" ■ series determination of bacteria in the ambient atmos-, · ^v phere (including a device for producing ·.

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and localization of a reproducible small sample volume).

Figure 2 shows a polarographic cell in greater detail with an oxygen membrane in combination with a second device for setting and localizing a reproducible small sample volume under measurement conditions.

Fig. 3 gives a schematic representation of a balanced Two-cell bacteria measuring instrument, in which the electrical output power of the two cells against each other switched and the differential current is measured.

Fig. 4 schematically illustrates an apparatus for enclosing a Reaction volume represents together with a device to selectively a sample and hydrogen peroxide solution to introduce.

Fig. 5 shows schematically another embodiment of the Construction according to Fig. 4.

Fig. 6 s.tellt schematically shows an arrangement with zero adjustment, in which individual reaction chambers are used with a double cell which forms a positive sealing point with the base plate of the housing.

Fig. 7 shows various exemplary configurations of the cathode, which the required ratio of length to illustrate width of exposed cathode surface.

In the arrangement for the determination of bacteria in the atmosphere According to FIG. 1, the bacteria from the atmosphere are thereby against the filter disk 11, which is attached to the filter holder 12 is hurled, that air is sucked in from the atmosphere through an impingement nozzle 13 of high volume by means of the pump l4

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will. After a certain period of operation, the filter holder 12 is removed from the housing 16 and a catalase-free one Liquid such as sterile water is added to the filter disc 11 and the wet filter then becomes one Exposed to ultrasonic energy by being in contact with the Ultrasonic transducer 17 is brought to the cell walls of the To tear bacteria apart, the transmitter 17 its energy from an ultrasonic generator 18 received. There can be other means of tearing apart the cell walls and releasing the cells soluble components thereof can be used, for example detergents or dissolving enzymes can be used. Through this dissolving process, the intracellular catalase enzyme effectively exposed. The types of bacteria in which a effective penetration of the undissolved bacterial cell wall Hydrogen peroxide can occur, this dissolution step can be omitted.

The filter holder 12 with the processed filter disk 11 held therein is then inserted into the chamber 19 of the housing 20, the filter disk 11 being placed in close contact with the polyethylene membrane 21 of the polar graphic membrane cell 22. Cell 22 is an electrolytic device for use in chemical analysis and is of the general type in construction as disclosed in U.S. Patent 2,913,386 to Clark, Jr. is described. The anode 23 and the cathode 24 of the cell 22 are electrically connected by a "trapped electrolyte ¹ - 'or by an electrolyte-forming substance 25 which is protected from turbulence and physically by the non-porous membrane 21 from the liquid sample to be examined. is isolated and electrically insulated, which is provided for oxygen, but not hydrogen peroxide, is permeable to the filler opening 26 in order, if desired, to allow the exchange of electrolyte Preferably, this opening 26 with a ground ^glass... locked cram the diaphragm 21 extends over the surface of the platinum cathode 2 ² I and rests firmly against it, the

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Cathode 21 embedded in a glass rod and held by it and wherein a specific cathode area at the distal end of the glass rod according to the improved construction the invention is exposed.

Oils show significant differences between the construction of the earlier polarographic cells (as they are in their typical Form disclosed by the device described in U.S. Patent 2,913,386), sifiü the configuration of the exposed Cathode and the provisions to ensure a seal between the polarographic cell and the structure of the Reaction volume. Both aspects are discussed in the below Description discussed in detail.

If desired, direct application can be used instead of the bacteria on the filter disk 11, the bacteria are introduced into a liquid by impact and the bacteria can then be transferred to a filter disc that the liquid is filtered through.

Before or after inserting the filter disk 11, dilute hydrogen peroxide (0.01 molar to 1 molar) is introduced into the chamber 2M. When bacterial catalase is present, the hydrogen peroxide (in oxygen and water) decomposes very quickly and the partial pressure of the oxygen in the hydrogen peroxide solution increases rapidly (in about 2-5 min.) To a value which corresponds to the amount of the catalase enzyme present ₍ The amount of oxygen diffusing through membrane 21 into cell 22 is linearly proportional to the oxygen partial pressure in the adjacent hydrogen peroxide solution, and the entering oxygen generates a cell current which is measured directly by microammeter 27. This measured current is blocked by the amplifier 28 and finally recorded on the connected recorder 29, a potentiometer recorder with a scale range of 1-10 millivolts.

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A platinum electrode (a wire with a diameter of 0.6 mm. Embedded in glass) is bent at the upper end and is essentially parallel to the membrane 21. The glass and>. a part of the metal are ground to a flat long _s · J thin cathode surface (about 0.06 mm by about 1 mm ₃ 8O) (about 0.002 inch by 0.0625 inch) freizulegen.'Diese cathode 2k is tightly covered with a 0.025 mm (0.001 inch) thick polyethylene membrane that retains the potassium chloride (0.3 to 3 molar) electrolyte in cell 22. Both the cathode 24 and the silver-silver chloride anode 23 are at dss \ - connected and ammeter 27 from a power source such as battery 31 v ier a predetermined electric potential at "le * j is applied. The simplified overall reaction is as follows: '

H e ~ + 1/2 O ₂ + H ₂ O \ 2-KCl +. Ag -> 2 AgCl ♦ 2 KOH + He "I

A relatively large volume of electrolyte is provided in the cell 22 in order to avoid the effects of changes in the electrolyte; reduce a mini space and cells of this type work up to j stable to a few weeks. The inherent sensitivity Γ the measuring device is at least 2.71 x 10 Aftp, / Ätmoephärö

the oxygen partial pressure. '

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The spontaneous decomposition of hydrogen peroxide was avoided by stabilizing the solution introduced through the small opening 30 with acetanillide, which was buffered in a slightly acidic manner (pH 6.8) with a 0.1 molar phosphate (pH) H buffer, "Dilute Hydrogen peroxide solutions can easily be brought into equilibrium are brought and kept on almost every given partial pressure, in which one continuously applies a thin layer exposing the solution to a gaseous environment which: has the desired partial pressure of oxygen, i.e. it is left open * " to the atmosphere. ·

The sensitivity of the cell 22 to the determination of the dissolved Oxygen content in the hydrogen peroxide solution is in principle determined both by the permeability characteristic of the ν Membrane 21 with respect to oxygen and by the dimensions of the

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Surface of the cathode 24, which corresponds to the membrane 21 is exposed to oxygen passing through. When looking at the top of the cathode structure, the electrode appears as a substantially elongated thin straight or curved surface having a length to width ratio of at least 25: 1 and preferably 100: 1, with your Width is less than about 0.127 mm (0.005 inches), and preferably less than about 0.051 mm (0.002 inches). Although preferably If polyethylene is used for the membrane 21, any membrane material can be twisted that can be used as a whole (non-porous) very thin membrane can be made for hydrogen peroxide is impermeable and has an oxygen permeability equal to or greater than 10 cnr / sec.

If it is desired to have very small numbers of bacteria (i.e. about 100 or less) or from other cells with the help of a detect a measurable increase in partial pressure, then it is critical that the volume of the hydrogen peroxide solution very Is kept small, preferably smaller than 0.1 cm .. A very effective means of maintaining such a reproducible small reaction volume »which is adjacent to the oxygen Electrode 23 can be attached is to use a small filter made of cellulose acetate or cellulose nitrate or a similar porous material for the Disk 11. In the device according to FIG. 1c, the filter disk 11 is positioned between the membrane 21 and covering the electrode 24 the oxygen-impermeable carrier body 28 inserted, which is attached immediately behind the filter 11 and it in the correct position in the chamber 19 holds.

The hydrogen peroxide solution is carefully injected through the small opening 32 into the chamber 19 so that organisms that are present on or in the filter disk 11, cannot be displaced from their position. One can get a similar result achieve that the hydrogen peroxide solution the soil

the chamber 19 before the introduction of the filter holder 12 and in,

Disk ll ^ covers the chamber 19, in which case the filter

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disk 11 during a very rapid immersion process gets soaked.

Once the filter disk 11 is wetted, the convective Mixing between the volume defined by the disk 11 and the surrounding hydrogen peroxide solution is effective limited, but the molecular diffusion into the filter disk 11 from the surrounding hydrogen peroxide solution remains about the same as in the case of a free liquid. Consequently is the longitudinal diffusion path for the released Oxygen gas to escape from the volume of the filter disc 11 relatively long, while the diffusion path to the oxygen electrode 24 is relatively short. Hence the by the presence of bacteria (or some other catalase. source) in the resultant hydrogen peroxide decomposition generated oxygen partial pressure effectively close to the oxygen sensitive Electrode 24 is retained within the filter disk volume and can develop to relatively high values build up. This high level of oversaturation with oxygen, which depends on the amount of catalase available in the area " between 0.01 to 1 atmosphere is maintained for a long period of time will.

Another arrangement for generating the reproducible small sample volume is shown in FIG. A filter disc 41 is inserted into a recess in the removable support body 42, which is in the working position to the Chamber 43 in which housing 46 is shown. This arrangement gives better isolation of the examined sample volume. The structure of the oxygen membrane cell 47 is essentially the same same as cell 22 except for the other device for supplying electrolyte (feed pipe 48). the Wasseratoffpero3; idlÖ8ung is via the filter holder 50 penetrating channel 49 carefully introduced into the chamber 43 ′. Although © in © corresponding device is not shown, e ^ hydrogen peroxide solution can instead in the chamber 43 before ι

909848 / 0SS4 BAD ORIGINAL

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Insertion of the filter and the support body ¹ 12 introduced

j .. The --Werden kl in dera reaction volume of the filter disc erseiigt © oxygen gas passes through membrane 51 oils therethrough and. After fts has entered cell Hj and arrives at cathode 52 and has absorbed electrons, a current is generated which is measured by the M-microammeter amplifier 53 with a low input impedance and calibrated by the potentiometer writer 54.

In order to illustrate the measurement sensitivity that can be achieved with the small, reproducible sample volume according to the invention (which is either a quantity of porous material or a positive reaction between catalase and hydrogen peroxide), a practical filter disk with a thickness of 150 microns is used and a diameter of 0.5 EA Coöer ® ± η®τ surface won O _s 196 cm ²⁾ is considered, the 'j * W | t 8 ° cavities% © athllt "the © © rubs a sample or solution volume of S ₉ 38 χ 10 "* & wr_ ia am? Filfeeraehelbe. Ea is the following Beiiehu ^ g %

N β 2.68 χ! Q ¹ Vv CpO-) (1)

In it means'

N is the number of oxygen molecules in a given Volume (V in cm) of the aqueous. Solvent,

the partial pressure of oxygen in atmospheres, which one is produced by these N-molecules,

the absorption coefficient for oxygen * This is the ^{volume of oxygen (reduced to O 0} C tmd 760 mm of mercury) that is absorbed by one cm of the test solution when the pressure of the oxygen without the water tension is 760 mm of mercury.

909846/0914

ORIGINAL

If one assumes the absorption coefficient A * for oxygen in dilute hydrogen peroxide solution at 20 ⁰ C to be 0.028, then one obtains:

N = 7.5 x 10 ¹⁷ V (p0 ₂ ) (2)

Assuming that of a given number (n) of bacteria, each of which has essentially the same number (a) of oxygen molecules generated in the cell during the reaction between catalase and hydrogen peroxide, a total of them is generated by N-oxygen molecules, then reads the above

™ relationship

na = ■ 7.5 x 10 ¹⁷ V (p0 ₂ ) (3)

As described in the article "Crystalline Bacterial Catalase" by D. Herbert and J. Pinsent, (Biochem. J. 43, 19 ^ 8) reported

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1.9 x 10 molecules per catalase molecule per second: Hydrogen peroxide decomposes and by any aerobic bacteria

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will be available, provided 10- ³ to 10 Katalasemoleküle. \

By running this reaction for 100 seconds, will

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the oxygen production per bacterium is greater than 10 molecules j

12 ■ '

Oxygen. At this value (10) for a and at a volume. f

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For a hydrogen peroxide ^{solution of 2.38 χ 10 J for} _s , the above equation (3) gives a value of 5> 6 x 10 "atmospheres of oxygen that is released per bacterium. This significant high oxygen partial pressure for a single bacterium | enables quantitative determination the presence of |

less than 100 bacteria in a liquid using the improved cell construction disclosed herein.

In Fig. 3 an arrangement for increasing the discrimination of the construction shown in Fig. 2 is shown schematically, in which two membrane oxygen cells 61, 62 are used. The electrical outputs of cells 6.1 and 62 are in the The electrical arrangement shown is connected in series against one another and the differential current is measured. The impact of everyone

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Parameters which affect cells 61 and 62 in the same way are canceled in that the potentiometer 63 and the variable resistor 64 are set so that the display on the recorder 65 is zero before the catalase determination is carried out. Thereafter, when the formation of oxygen gas is determined at the same time, the output currents from cells 61 and 62 reflect the difference between the output currents from these two cells. Although cells 61, 62 are shown in communicating connection with the same chamber 74, separate chambers (one for each oxygen detection cell) can be used provided there is good thermal contact between the cells to maintain identical temperature conditions.

In this way "those variables (temperature and absolute level of the oxygen partial pressure in the liquid) which affect both cells in the same way can be canceled. Furthermore, this arrangement enables differential measurements to be carried out in which two identically collected bacterial samples which are the same For example, the sample isolated on the filter disk 66 can be treated by heating or by adding chemicals which specifically inhibit the catalase, in order to inactivate the catalase content, while the sample isolated on the filter disk 67 is untreated Assuming that the deactivator does not inactivate other hydrogen peroxide decomposition catalytics (e.g. iron oxides) that may be present, then these catalyst agents will decompose an equal amount of hydrogen peroxide in both cells n 6l and 62. These output currents are equal to each other • aüs _; while the deactivated catalase does not produce hydrogen peroxide decomposition to compensate for the hydrogen peroxide decomposition of the untreated catalase. This provides a differential output which can be measured by the microvoltmeter 68 and recorded by the recorder 65.

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In the arrangement of Pig. 3, the filter disks 66 and 67 are positively pressed against the membranes 69 and 70 of the oxygen cells 61 and 62 by the Peder loading of the filter holders 71 , 72. The spring-loaded filter holders 71 and 72 are attached to a mounting plate 73 which covers the chamber 64 in the housing 76. The walls of the housing part 76 can be made of metal or plastic; however, they are preferably made of transparent material, for example plexiglass. The hydrogen peroxide solution is introduced into the chamber 74 either before the filter is inserted or through the channel 77 from a hydrogen peroxide reservoir (not shown).

The oxygen cells of the invention are designed ₃ so that the shape of the upper portion promotes the seal between the oxygen cell and the housing. In FIG. 2, the upper is formed, for example with the membrane-covered portion 56 of the cell 47 as a truncated cone and tightly fitted in a matching recess 57, which check the bottom of the housing 46 passes through.

By using balanced oxygen cells, As shown in Fig. 3, there is an improved • measurability of the oxygen partial pressure, since with this arrangement becomes possible to measure the output current with higher sensitivity. For example, a single one supplies oxygen cell of the type described here at partial pressure of oxygen of the free atmosphere (0.21 atmospheres) has an output current of about 5.7 x 10 amps. When using a single Cell, the sensitivity available for current measurement is necessarily limited to this order of magnitude. Through the When two cells are connected against each other, the usable sensitivity is limited only by the noise level of the system. Instead of the second cell of FIG. 3, a voltage source be used for a constant counter voltage; these Arrangement would, however, only give the possibility of the temperature and the absolute value of the oxygen partial pressure only partially to compensate.

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Experiments with undissolved aerobically grown E.coil have resulted in a values in the range of 5 x 10 ¹⁰ to 2 χ ΙΟ ¹² molecules of oxygen generated in 2 minutes per bacterium using a 0.1 molar hydrogen peroxide solution. Other bacteria can be evaluated similarly and there will no doubt be differences between the different bacteria. For example, the catalase content of E. coli is considered to be relatively low. A value of a greater than 10 molecules of oxygen per undissolved bacterium would render the use of dissolving enzymes or ultrasonic energy unnecessary unless it is necessary to detect a very small number (less than 100) of bacteria. However, for a values of less than 10 molecules of oxygen per bacterium, resolution is preferred and in this case the method of collecting the bacteria on a filter disc makes the application of ultrasonic energy (as described in connection with FIG. 1) particularly convenient. The moistened filter disc only needs to be brought into contact with a source of ultrasound energy for a short time in order to obtain excellent energy coupling to the bacteria. With this method, only a short treatment time is required for an effective dissolution process.

4 shows yet another arrangement of a sample reaction volume shown in combination with an oxygen measuring cell. The reaction volume 81, which is for the catalase water Peroxide reaction provided in the total volume is generally 1 cubic centimeter or greater in volume. However can this arrangement can also be conveniently applied to highly concentrated samples to which hydrogen peroxide solution has been added shall be. In such cases, higher sensitivity is desired and the volume 81 should then be 0.1 cm or less be. The housing 82 is detachably fastened to the base plate 83 by fastening means such as the screws 8 ^ and the Reaction volume 8l is connected to the oxygen cell 86 via the membrane 87, which is part of the wall surface which defines the volume 31. The ones with the inside of the

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Volume 81 communicating valve ports (88, 89, 91) J. are shown. These respective connections are used to enable: (a) the supply of hydrogen peroxide solution (for both the catalase-hydrogen-peroxide reaction) and for flushing the volume 81 between measurements) .sus dem Hydrogen peroxide solution supply 92, (b) for the introduction, of sample liquid (for example urine, oil tap water, etc.) of the sample queue 93 and (c) for the elimination of. Air and for flushing of reaction products from the overflow pipe 94 ... As, suitable Means for introducing the sample liquid and the Injection syringes are shown with hydrogen peroxide solution. · 'I; The sealing of the volume 81 is achieved by the deformable ... Ring seal 96 guaranteed. ■

In the construction shown in FIG. 5, the reaction sample volume 101 is formed by the housing 102, through which the valve connections 103 * 104, 106 and 107 and the small shaft 108 of the stirrer pass. This arrangement is primarily intended to be used for reactions ί between kätalase and hydrogen peroxide with the entire sample; volume, as when measuring urine samples. Although for convenience of illustration the device for moving the liquid is shown as a combination of a propeller and an axle, it is preferred to use a magnetic stirrer which does not require penetration of the wall of the housing 102. Detachably therebetween, e.g. , connected housing 102 and the base plate 109 is arranged deformable sealing material 109. The hydrogen peroxide solution is introduced into the volume 101 from the source 113 as required and the sample liquid to be examined for its catalase activity is introduced into the volume 101 from the source "" 114 as required The hydrogen peroxide solution volume added to the sample liquid is preferably less than 10 percent by volume of the sample volume: however, this is not an indispensable condition. The washing liquid is forced into the housing "V" ^! 102 as required ^{r brought in} from the source 'Il6 "; all sources'.'; 113, 114 and Ho are conveniently used as injectors

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indicated. The liquids introduced into the reaction sample volume 101 are displaced by the stirrer 117 as required moved- to reduce or eliminate concentration gradients in the liquid and the overflow 104 is used, to remove air and while flushing the Eject the contents of the volume 101. The one with a membrane

covered oxygen cell 118 is in the same way like the cell 86 with respect to the volume 81 (Fig. 4) on the Reaction sample volume 101 arranged.

The structural configurations according to FIGS. 4 and 5 are special useful in determining generally large catalase contents in liquids, such as in examinations for infections of the urinary organs.

It is also proposed to apply this invention to the automatic to apply quantitative determination of the catalase content, for example by using a moving belt (or if desired a continuous belt) showing the order of the stages of sampling, dissolving, the Reaction development and measurement at successive stations. The tape could be a layer of a thin one porous medium, such as cellulose acetate or agar, which are placed on a surface with low oxygen permeability, for example, a relatively thick (i.e., thicker than 0.5 mm) (20 mils) plastic material. Although continuous movement of the belt is possible, incremental movement of the belt is more practical. For example would be a more discreet one when determining the bacterial content in the atmosphere in a 5-minute cycle Part of the porous medium is first sampled in a first place over a period of about 2 minutes Subjected to air impact. This discrete part of the The tape would then be moved forward to the sample, dissolving for half a minute using ultrasonic energy to submit at a second station. Eventually it would

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Belt to be moved to a third station where the initiation, the measurement and recording and / or transmission of the results of the reaction take place in accordance with this invention would.

The inherent sensitivity of the reaction between catalase and hydrogen peroxide employed in this invention can be further enhanced by using, for example, a small thin disk of agar or similar material ₃ which can be selectively liquidated as the medium for the initial sampling . Sampling is originally carried out on a given agar disc. This first agar disk is then heated in the liquid state and the resulting liquid is filtered through a fibrous filter disk of smaller cross-sectional area than the agar disk, thereby giving a further concentration of the collected bacteria. For example, an agar disk originally used for sampling with a diameter of 25 mm would provide a concentration of 25: by subsequently heating the agar and filtering the resulting liquid through a fibrous filter disk with a diameter of 5 mm.

6 shows a double cell 21 for oxygen determination in a single housing 122 for electrical counter-connection in series. It is _s provided a pair of adjacent reaction chambers 123 124, each with one of the cathode 126 are in a connection to the measurement of oxygen 127th The cathodes 126, 127 are electrically connected to a "common anode 128" (which is connected to the positive pole of the battery 128) through a common electrolyte reservoir 129. The ring 131 which can be screwed onto the base plate 132 presses the cell 121 tightly against the base plate 132 the wells 133, 134. The base plate 132 is preferably made of a material that is a good conductor of heat in order to keep the two cathode sections of the cell 121 at the same temperature, the walls 136 and the top

909846/0964

They are preferably made of a transparent material that is resistant to oxidation.

, Pas reservoir 138 absorbs the liquid test sample, which can be pumped through the lines surrounded with valves lines simultaneously by the pump 13t into the two reaction chambers 12-3 "124th The reservoir I4l contains the hydrogen peroxide solution, which can be pumped into the two reaction chambers 123 »124 simultaneously by the pump 142 via the lines provided with valves. The pipelines 143 with valves 144 serve as outlets for the two reaction chambers 123 and 124. The reservoir 146 contains a catalase-inhibiting substance, for example 2% sulfuric acid, which can be introduced into the chamber 123 by the pump 147 as required.

In operation, equal parts of a urine sample are placed in reaction chambers 123 and 124 so that they fill most of the volume of the chambers. To suppress the ■■ catalase activity in the reaction chamber * 123, a small amount of the inhibitor is introduced and the potentiometer

148 is set so that there is a zero display on the recorder

149 receives, the electrical outputs of the cathodes 126, 127 are connected in series against one another. Then there the measured increase in oxygen partial pressure is exactly that Catalase activity, which is still compensated with regard to the temperature and the oxygen blank value,-again when the Hydrogen peroxide solution into the reaction chambers at the same time 123 and 124! Is introduced.

As stated above, one aspect of this invention of considerable importance is the increased sensitivity achieved by the use of a long; thin exposed cathode area, i.e. an area with a ratio 'from length to width of at least * 25:' 1 is offered. In FIGS. 7a, 7b and 7c show examples of various useful configurations. These are views of the surface of a cathode embedded in glass 151 voff ^ erc oxygen-

9 0984 6/0 9-64- - ° ^ ^

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permeable membrane seen from. A method of manufacture. Such cathode surfaces consists in that an end section 152 ^{v of} the upwardly extending cathode wire (for example platinum, gold) is bent at a right angle. The configurations shown in FIGS. 7b, 7c are obtained when these ends are bent in an arc, in a zigzag shape or in some other shape and then the entire wire is embedded in glass. A metal surface of long, narrow shape (153, 154, 156) is then exposed by grinding off the glass and part of the metal. 1-

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^r In those cases in which a time delay between the sampling and the measurement can be accepted, the bacteria samples collected on a filter disc can be impregnated with nutrients, for example to promote the growth of the bacteria present therein or to achieve the an inhibitor can be added to the opposite effect. As an application of this possibility, for example one of two samples collected at the same time (or two parts of the same sample) can be treated by the application of antibiotics in such a way that the growth of their bacteria is inhibited. The growth of the bacteria in the untreated sample will increase the catalase activity and a comparison between the catalase activities of the two samples will then give a quick indication of the effectiveness of the antibiotic attack on the bacteria. The same treatment, of course, also gives a measure of the viability of the collected samples. A high reading of differential output current indicates high sensitivity of the bacteria to the antibiotic and at the same time indicates low viability. Inhibitors and agents which 'selectively inhibit or promote a particular species of bacteria can also be used to identify bacteria. For the cultivation of colonies in conventional microbiological culture techniques, a long time (at least 2k hours) is required for the growth of the organisms. Because

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the high system sensitivity available with the oxygen membrane cell and the small reaction volume according to the invention "are * will these long growing times for these experiments for I, viable, sensitivity or for identification of bacteria not needed and a single duplication Bacterial growth over a period of 20 to £> Q minutes should be easily detectable and sufficient to observe the effect of the inhibition or growth retardation. .

In summary, the above devices and methods for determination the catalase activity has been demonstrated, which quantitatively and quickly directly in a hydrogen peroxide solution Measure the increase in oxygen partial pressure, the inherent catalase content of various animal cells causes the rapid breakdown of hydrogen peroxide into oxygen and water. The disclosed device gives the possibility for dynamic and continuous observation of the catalase-hydrogen peroxide reaction and enables quantitative measurement of catalase activity.

A shape of the cathodes was made, which is one significantly higher output current for a given concentration gradient of oxygen in a hydrogen peroxide solution.

Devices have been developed and disclosed for limiting the effective sample volume of hydrogen peroxide solution and Sample (less than about 0.1 cm) in the measurement duct. The simplicity of the devices disclosed and the speed with which the quantitative catalase activity can be measured and recorded can make the present invention a very useful tool in the clinic. For example, it is highly desirable the ability to have cheap, rapid mass screening to diagnose urinary tract infections, before symptoms of infection appear. When an examination of a patient's urine by the method and with a

909 8 4 6/0964

BATH

; Apparatus according to the invention has significant catalase activity shows, then it is only necessary to make more definite

; conduct tests to determine whether the large catalase -! ■ activity is due to the presence of bacteria, |. or whether an unusual destruction of catalase Cells occurs or within the patient's body.

^! is kicking.

.

If an infection of the urinary excretory organs is already diagnosed bacteria and other cells present in the urine are first filtered out. Then a measurement is made to determine the catalase activity of the filtered urine. If this measurement is positive, it is proof for the destruction of katalasehal- in the body. term cells (for example due to inflammation) is present.

An equilibrium double cell arrangement has also been specified which provides a more sensitive measurement of the Change in the oxygen partial pressure in a hydrogen peroxide solution results in the scattering as a result of spontaneous Decomposition of hydrogen peroxide and of temperature effects are reversed. This is especially important because the Temperature sensitivity of the disclosed oxygen measuring cells is about £ 5 / ° C.

Likewise, the use of a pair of oxygen membrane cells connected in series with one another and the determination of oxygen entering the cell from various fluids, one of which is from catalase activity free reference liquid, e.g. water, ist.-Indsm prior to adding the hydrogen peroxide solution to the sample liquid and to the reference liquid, cells and the liquid can be brought to the same temperature conditions and then a potentiometer recorder to zero is calibrated, both the temperature and the reactive oxygen content are measured eliminated as fluctuations.

9098 AS / 0964 BAD ORIGINAL

Furthermore, the arrangement with balanced cells allows the measurement of the catalase activity in the presence of other possible agents for the hydrogen peroxide decomposition, which in the process of sampling may have been collected by performing the measurement on two samples collected at the same time one of which is used to selectively inactivate the catalase without affecting the activity of other hydrogen peroxide catalysts is treated in the sample.

■

Se becomes a method of separating bacteria from the atmosphere described by impact effect »in which the bacteria are applied directly to a small filter and at a sufficient number of bacteria can easily be collected in a short period of time to be present in the Hydrogen peroxide system to provide a measurable indication. By the use of a small filter disk can prevent bacteria from becoming infected Liquids can conveniently be sampled, and also offers the option of concentrating bacteria

Iί ", BUr Introduction to a Small Reaction Volume.

The reaction between catalase and hydrogen peroxide is very specific, but a hydrogen peroxide solution per se need not be used. The hydrogen peroxide can be put in place by the addition of agents such as NaBO ₂ . 3H ₂ O

• H ₂ O ₂ I sodium perborate can be formed into water or the liquid sample.

909846/0964 BAD

Claims (1)

EXPECTATIONS: I) / device for rapid quantitative measurement dta at Reaction generated gas, g e k β η η ε e rejects by,. the combination of: .- "-., a housing (20,46,82,102), a base plate (83, Hl) ₃ which supports the housing and encloses a volume (19,43,81,101) with the housing, a recess (5T) in the base plate (83, in), which is in connection with the volume _Ai (19,43,81,101), a polarographic membrane element (22,47,86,118), which comprises an anode (23) and a cathode {2k, 52 "> which is connected via an electrolyte ( 25) are connected to one another, the membrane cell having a non-porous gas-permeable membrane (21,51,87) as part of its outer wall, a first device (p, 49,88,103t • 106) for the regulated supply of a first liquid in the volume, a second device (89,102) Eur! ** th supply of a second liquid in the volume UnA of a device (91,94,104) for venting the volume in order to let the air out of it when the liquid is poured into the volume. 2) Device according to "Claim 1, since you know that the non-porous membrane (21, 51, 87) is impermeable to hydrogen peroxide and has an oxygen permeability of at least about 10 years / eec . ·. ■ "■■" ■ " 3) Device according to claims 1 or 2 * d ad u r ο h ge.kenn ζ cal η et, that the electrolyte is exposed to the cathode area (153.15 ¹ J, 156), a ratio of. 'Has length to width greater than about 25: 1 and has a maximum width of about 0.127 tnm (0.005 inches) 1k I * 9098A6 / 0964 BATH 4) Device according to claims 1 to 3, characterized characterized in that the part of the oxygen measuring cell (22,47,86,118), which in the recess (57.) protrudes, has the general shape of a truncated cone, and that the recess (57) has an inner surface into which this fits in the form of a tight fit. 5) Device according to claims 1 to 4, characterized marked that it also contains a combination of the following features: a filter support element attached to the housing (20,46,82,102) (28,50), which protrudes into the housing in such a way that a carrier
End part of the filter / · is arranged adjacent to the membrane (21, 51, 87), a filter (11, .4l), which is arranged between this part of the filter support element and the membrane, and a device for the controlled supply of a liquid into the housing in contact with the filter disc. 6) Device according to claim 5 * characterized in that that the volume of the filter disc (11,41) is less than about 0.1 cm. 7) Device according to claims 5 or 6, characterized characterized in that the filter disc (11, 41) in a recess in the surface of the part of the filter support element (28,50) which is attached to the Membrane part (21,51,87) is adjacent. 8) Device according to claims 1 to J _{3 j;} because dur marked eh ch η et that they also the combination , includes the following features:. -V .. Device (27,28,29,53,54) for measuring and recording changes in the electrical characteristics of the Oxygen measuring cell (22,47,86,118;) with an impressed electrical potential, these changes due to 9D9846 / Q984 ■ Τ-— r —— j ~ - the ionization of the oxygen-permeable part of the membrane (21,51,87) and the subsequent chemical reaction between the oxygen ions and the electrolyte will. 9) device for rapid quantitative measurement of catalase activity, d a d u r c h g e k e η η ζ e i c h η e t j that it comprises the combination of the following features: a housing (74,123,124) with a base plate (132), a pair of separate recesses (133,134) that penetrate this base plate and with the interior of the Housing connected,, a pair of oxygen measuring cells (61,62.), which respectively contain an anode and a cathode, which are through a Electrolytes are electrically connected to each other, and have an outer wall part adjoining the cathode and consisting of a non-porous membrane (69,70), which is impermeable to hydrogen peroxide and an oxygen permeability of at least about 10 cm / sec. having, wherein one of the oxygen measuring cells, which protrudes into the first recess and the other oxygen measuring cells, which protrudes into the second recess, together with the membrane parts of the wall of the measuring cells, this recess w seal rules and the bottom portion in liquid-tight finish, a pair of Filterträgerelementeri (71,72), which protrude into the interior of the housing, wherein an end portion of the first filter element adjacent to the membrane portion of the first oxygen sensing cell and an end portion of the second filter support member adjacent to the diaphragm portion of the other Oxygen measuring cell is arranged, a first filter disk which is arranged between the part of the first filter support element and the membrane part of one of the oxygen measuring cells and a second filter disk (66,67) which is arranged between the part of the second filter support element and the membrane part of the other oxygen measuring cell,. . · -..- Device (77) for admitting a liquid into the housing • (74) in contact with both filter disks (66,67) and one 909846/0964 BATH ORIGINAL j device for electrically connecting this pair of Oxygen measuring cells in series and with opposite polarity to one another. 10) device naoh claim 9, .d As a result g e -. : markzeio h * n β t that the parts of the oxygen measuring '· Cells which protrude into the recesses, in general are designed in the form of a truncated cone, and that these recesses each have an inner surface, which accommodate these ^ in the form of a tight fit * 11) device for rapid quantitative measurement of the in; a reaction formed gas, gekennzeichj net by the combination of the following features: a pair of ton chambers (123,124), each of these chambers having a base plate (132) and a recess (133 * 134). has, which passes through this base plate and are in connection with the interior of the housing, first and second polarographic membrane cells (121), which each comprise an anode (128) and a cathode (126,127), which are connected to one another via an electrolyte (129) in Are connected, and each part of their outer wall has a non-porous permeable membrane, and wherein the first polarographic membrane cell (121) protrudes into the recess (133, 134) through the base plate of one of the chambers (123, 124) y \ , and the second polarographic membrane cell! (ISIi protrudes into the recess (133 * 134) through the round plate of the other chambers (123 »124) and the cells ^: close these recesses and seal the base plate regulated supply of liquid from a common ^r ; Reservoir (138, 14l) in each of these chambers (123,124), one '' Device for the electrical connection of the membrane cells ■ '(121) in series and with opposite polarity to one another and, a device (149) for measuring and recording the resulting voltage difference between the polarographic membrane ■ ^f . cells. . · 909846/0964 BADORIQINAi 12) Method of operating the devices according to claim 2 to 11 for the clinical measurement of the presence of catalase-containing cells in body fluids, thereby iv characterized in that an amount of body fluid is withdrawn, part of this amount Her Body fluid is introduced into a closed volume to partially fill the volume, an amount of hydrogen peroxide is added to the fluid in the volume that is sufficient to completely fill the volume, and that the increase in the partial pressure of oxygen, which enters the hydrogen peroxide solution in a preselected period of time, with the help of one or two polarographic Cells are measured and recorded with an oxygen-permeable membrane. 13), method according to claim 12, characterized in that the volume of the added hydrogen peroxide solution does not exceed 10% of the volume of the body fluid. .. .J A method according to claims 12 or 13, characterized in that the body fluid consists of two substantially identical separate parts of the material to be examined for catalase activity, and that a sample of each of these parts is introduced into a separate closed volume and the catalase content of each of these parts is affected differently, and then the same hydrogen peroxide solution in each of these volumes is added together to complete filling of the volume of the containing therein the sample liquid and the difference in oxygen partial _t "pressure, which auftri1fc in the Wasserstoffperoxidlößung in the separate reproducible volume, while a preselected period of time is measured with the help of polarographic cells with oxygen membranes, which are connected in series and with opposite polarity to one another. 9098A6 / 0964 BAOOFHGINAt. 15) Method according to claim 12 to 14, characterized ge indicates that the increase in the partial pressure of oxygen is continuously measured electrochemically in the liquid-filled volume and this Measurement is recorded continuously for visual observation. "■ · ■" _ 16) Method according to claims 12 to 15 _3, characterized in that a pair of polarographic cells with oxygen membrane are arranged in connection with a pair of reaction volumes, each of these cells being with one of the reaction volumes in a liquid-tight connection and these cells have opposite polarity and are connected in series and the output current of the cells is measured. 909846/0984 . - 30 -τ blank page