GB2063911A - Measurement of increase of bacteria - Google Patents
Measurement of increase of bacteria Download PDFInfo
- Publication number
- GB2063911A GB2063911A GB8034498A GB8034498A GB2063911A GB 2063911 A GB2063911 A GB 2063911A GB 8034498 A GB8034498 A GB 8034498A GB 8034498 A GB8034498 A GB 8034498A GB 2063911 A GB2063911 A GB 2063911A
- Authority
- GB
- United Kingdom
- Prior art keywords
- bacteria
- nutrient medium
- potential
- increase
- electrode
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 57
- 238000005259 measurement Methods 0.000 title claims description 12
- 235000015097 nutrients Nutrition 0.000 claims abstract description 36
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 230000033116 oxidation-reduction process Effects 0.000 claims abstract description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims description 27
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 14
- 239000010936 titanium Substances 0.000 claims description 8
- 229910052697 platinum Inorganic materials 0.000 claims description 6
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims description 3
- 229910052719 titanium Inorganic materials 0.000 claims description 3
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 claims description 2
- 229910052721 tungsten Inorganic materials 0.000 claims description 2
- 239000010937 tungsten Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 7
- 238000012216 screening Methods 0.000 abstract description 3
- 238000011835 investigation Methods 0.000 abstract description 2
- 241000894007 species Species 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 8
- 102000001554 Hemoglobins Human genes 0.000 description 7
- 108010054147 Hemoglobins Proteins 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 6
- 230000000721 bacterilogical effect Effects 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 229910002462 C-Pt Inorganic materials 0.000 description 3
- 230000002411 adverse Effects 0.000 description 3
- 229910052709 silver Inorganic materials 0.000 description 3
- 239000004332 silver Substances 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 229940075397 calomel Drugs 0.000 description 2
- 238000012505 colouration Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical compound Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 229910021397 glassy carbon Inorganic materials 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 239000002296 pyrolytic carbon Substances 0.000 description 1
- -1 silver ions Chemical class 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
Abstract
The degree of increase of bacteria is detected as a change in oxidation-reduction potential of a liquid nutrient medium inoculated with bacteria. This change in potential is measured as a potential difference between a carbon electrode and a metallic electrode. Identification of the population and species of bacteria initially present in the liquid nutrient medium, the screening of bacteria present in samples, and investigation of the resistance of bacteria to medicines can be conducted on the basis of the time required until the potential difference changes markedly. Curves 2 and 3 of the drawing show the variation of the potential difference with respect to time. The abrupt rises in potential difference correspond to the concentration of bacteria reaching a certain level. <IMAGE>
Description
SPECIFICATION
Measurement of increase of bacteria
The present invention relates to a method for
measuring the degreee of increase of bacteria
inoculated in or on a liquid nutrient medium. More
particularly, the present invention relates to a
method for measuring the degree of increase of bacteria by inserting into a liquid nutrient medium
a carbon electrode and a metallic electrode neither
of which is adversely affected by the liquid
nutrient medium nor affects the liquid nutrient
medium, whereby a change in oxidation-reduction
potential of the liquid nutrient medium in the
course of multiplication of the bacteria is detected
as a change in potential between the two
electrodes.
Presently, in clinical bacteriological
examination, such as tests of sensitivity to
medicines, discriminations between inactive or
dead bacteria and active bacteria, estimation of
the medium efficiency through bioassay,
identification of bacteria and screening of bacteria
in urine, there are eagerly desired, in addition to
accuracy of measurement, ease and speed of
operation and, further, automation of
measurement. Advance in this direction is greatly
desired especially with regard to testing sensitivity
to medicines in developing new medicines against
recently generated bacteria resistant to previous
medicines.
Generally speaking, bacteriological examination
can be made most accurate by microscopically
observing the bacteria desired to be tested, which
are inoculated in or on a nutrient medium and
allowed to increase. This technique takes a lot of
time for increasing bacteria, requires patience and
skill and attentiveness for the measurement, and
is extremely inefficient. In practice, it consumes
many hours in measuring a large quantity of
samples.
For this reason, there has been developed
another technique, for the purpose of automation
in the field of the bacteriological examination,
which involves two methods for measuring a
change either in the degree of turbidity or in the
electric conductivity of a nutrient medium
inoculated with bacteria. One method is the
turbidity method. The other method is the
impedance method. The former method makes
use of the phenomenon that bacteria inoculated in
or on a liquid nutrient medium increase
logarithmically with time to make the medium
turbid, and the degree of turbidity of the nutrient
medium is measured by the use of scattered or
transmitted light.In the latter, impedance method,
a change in electric conductivity of a liquid
nutrient medium, which change is caused by a
metabolic product being formed in the course of
increase of the bacteria is measured by the use of
impedance bridge to determine the degree of
increase of the bacteria.
Conventional methods as mentioned above,
however, have several defects as follows:
In the turbidity method, it is essential to mix the nutrient medium by stirring, e.g. with the help of a vibrator, if the turbidity is non-uniform dependent upon strains of bacteria. Sometimes it becomes also troublesome to maintain and control the uniform turbidity of the medium because there must be applied a number of optical systems.
Further, in detecting the population of such anaerobic bacteria which increase slowly, it takes a long detecting time, involving on occasion the occurrence of measuring errors because of inactive or dead bacteria being simultaneously detected.
On the other hand, in the impedance method, the measured values are affected by the areas of electrodes, the distance between the electrodes, and the intensity and frequency of the electric current applied to the liquid nutrient medium at the time of measurement. Consequently the measured values are open to errors as a function of the amount of samples at the time of the measurement, the fluctuation of the electrodes, and a change in electric current. The durability of electrodes also comes into question.
Aside from this, in order to meet the desire to make quick the test of the sensitivity to medicines in the field of bacteriological examination, there has been developed a method of measuring the degree of bacteria increase based on utilizing the reducibility of hemoglobin. The principle of this measuring method consists in judging when and how the oxidized form of hemoglobin contained in the liquid nutrient medium changes into the reduced form of hemoglobin, and therewith the colour of the liquid nutrient medium turns from scarlet to dark red. Since the reduction of hemoglobin takes place owing to the drop of oxidation-reduction potential of a solution of the hemoglobin, it can be envisaged that the reduction of hemoglobin will be brought about by the drop of oxidation-reduction potential.
Taking into consideration the above mentioned prior art, the present inventors conducted various kinds of experiments based on the finding that the oxidation-reduction potential in the liquid nutrient medium changes with the increase of bacteria and can be measured as a potential change between two solid electrodes. As the result, they made the present invention.
The present invention enables the degree of increase of bacteria to be measured without being affected by the colouration or turbidity of a nutrient medium, the size and form of electrodes, the presence of dead bacteria and other factors.
The method of the invention comprises inserting into a liquid nutrient medium inoculated with bacteria a carbon electrode and a metallic electrode, neither of which is adversely affected by the liquid nutrient medium nor influences it, and a change in oxidation-reduction potential produced between the electrodes is measured.
The present invention also provides an apparatus which is simple and stable in construction and, if desired, can be made automatic on the basis of solid electrodes being used therein.
The invention is further described below with reference to the accompanying drawing.
The accompanying drawing shows typically three different increase curves of Es. coil which were measured by the method according to the present invention. In this example, as a nutrient medium was used BHl bouillon, and as electrodes were employed a carbon electrode at the detecting side and a platinum electrode at the reference side. Curves 1 to 3 represent the following cases, respectively:
In the case of curve 1 as a blank test was added 0.2% of NaN3; in the case of curve 2 was inoculated 2.107 of Es. call in or on 45 ml of nutrient medium; and in the case of curve 3 was inoculated 2.106ofEs. coil in or on 45 ml of nutrient medium.
The present invention is not described more particularly. First of all, the present inventors started from the surmise that the reduction of hemoglobin contained in a liquid nutrient medium inoculated with bacteria must be attributable to the drop of oxidation-reduction potential in the nutrient medium. and then arrived at the thought that the degree of increase of bacteria may be rapidly measurable by directly measuring the fluctuation in the said oxidation-reduction potential. In this manner, their investigation began.
As for the measurement of the oxidationreduction potential, first a platinum electrode as the indicating electrode is combined with a half cell such as a hydrogen electrode, a Calomel electrode or a silver chloride electrode as the counter electrode, and thereafter the potential between them was measured as a rule. However, these counter electrodes are necessarily constructed in a complicated form so as to be aerated and have liquid junctions, as a consequence of which there is a danger of disturbing the characteristics of the nutrient medium used for cultivation of bacteria, and also there occur various kinds of controversial structural and operational problems such as the instrusion of bacteria into liquid junctions at the time of use for many hours, difficulty of washing or sterilization at the time of being reused and so on.
In consideration of these circumstances, the present inventors attempted to measure the oxidation-reduction potential by using varieties of combinations of solid electrodes such as platinum
(Pt), silver (Ag), carbon (C), tangsten (W) and titanium (Ti), whereby simplification in construction should be expected.
In order to investigate fundamentally, the present inventors measured the changes in
electric potential of 1 M ferric chloride solution and a liquid nutrient medium (BHI bouillon) at the time when 0.1 M of ascorbic acid was added to them to
be reduced. The following results were obtained:
In ferric chloride, the potential difference
increased more greatly at the combination of
Pt-Ag by 35 mV, at C-Ag by 167 mV, at C-W by 65 mV, at C-Ti by 120 mV, at C-Pt by 114 mV, at Pt-Ti by 35 mV, and at Ti-Ag by O mV, respectively, as compared with the case of ascorbic acid not having been added; while, in BHl bouillon, the potential difference increased more greatly at C-Ag by 165 mV, C-Ti by 57 mV, at C-Pt by 216 mV, and at C-W by 124 mV, respectively.
Next, in surveying the stability of each pair of electrodes, when the stability of electric potential at each combination of electrodes was measured with respect to the BHl bouillon to which 0.2% of sodium azide was added as antiseptic, it was shown that the potential of every pair of electrodes was substantially stable after the lapse of 30 minutes (Curve 1 in the drawing).
The change in electric potential owing to the increase of bacteria was displayed in the two-step wave which rose slightly with the start of the bacteria increase and after exhibiting stability for a short time abruptly changed describing a steep curve (that is, the potential difference increased), as shown in curves 2 and 3 in the drawing where are represented the changes in electrical potential at C-Pt, for example, obtained when Es. coli is inoculated into 45 ml of BHl bouillon and cultivated at 370C. To be exact, there is not sometimes without a case where the abovementioned waves might descend for a while before the abrupt change.
In addition, if the population of inoculated bacteria are reduced to 1/10 to 1/100, for example, the commencement time of the change is delayed respectively depending on the ratio of the reduction, but there are obtained curves of similar shape to curve 2 or 3 in the drawing.
However, there is no noticeable difference in each case in respect of the degree of change in electric potential, which is about 1 50 millivolts.
The fact that the potential difference between electrodes changes markedly in such a way at a time in the advanced stage of the degree of bacteria increase can be explained as corresponding to the phenomenon that the electric potential changes suddenly when, usually in the oxidation-reduction system, either the oxidized form of the substance or the reduced form of the substance becomes very small in amount. The number of bacteria at the point of time when the potential difference changes markedly can be regarded as almost constant, supposing that strain of bacteria and species of nutrient medium or electrodes are specified in advance. Accordingly, when this number has been counted by microscopic observation, it is possible to find the time during which the bacteria initially inoculated in or on a nutrient medium at the start have increased to the above-mentioned number (the population of the bacteria at the point of time when the potential difference changes markedly).
Conversely, if the number of original bacteria and the time required for the bacteria to increase up to above-mentioned number have been determined beforehand in a suitable range of cases, then the number of bacteria initially inoculated can be determined easily and quickly from the time required for the bacteria to increase up to the abovementioned number.
It is also possible to identify the bacteria on the basis of the form of the potential difference curve, namely, the bacteria increase curve, and further to make not only a particular bacteriological examination but also the gross volume measurement (screening) of the bacteria which are present in test samples such as urine.
It is possible to conduct the test of the sensitivity to medicines (e.g. pharmaceutical compounds) of bacteria quickly and precisely on the basis of the degree of time-lag from the aforesaid reference time. In this connection, however, the efficiency of medical supplies can be measured simply by dividing one test sample into two parts, one part being left intact and the other being given a certain medicine, and by comparing the different times required for both parts separately to reach the point of time when the potential difference between them change markedly. In doing so, it becomes possible to decide as to the dosage of medicine which sould be prescribed for a patient.
Now referring to the various combinations of electrodes to be used in the present invention, it was ascertained as apparent from the above described experimental results, that the change in potential of the one combination which used the carbon electrode as one of the pair of electrodes is greater than that of the other combinations which use exclusively metallic electrodes (as both electrodes), and consequently the first-named combination of electrodes has a better measuring sensitivity. Incidentally, the use of a silver (Ag) electrode led to the increase of bacteria being restrained. This was thought to have been caused by the sterilizing effect of silver ions produced by the dissolution of the electrode in the nutrient medium. This is the reason why the Ag electrode was not able to be used in the above measurement.
Consequently, it is desirable to employ always a carbon (C) electrode as one of a pair of electrodes, and, as the other electrode, an electrode of a stable metal such as is not adversely affected by the nutrient medium and does not influence it, for example, platinum (Pt), tungsten (W) or titanium (Ti). Among these, platinum is best from the standpoint of corrosion resistance, but if viewed from the angle of economy or the problem of the combination with the nutrient medium, the other metals also can of course be used effectively. In this connection, though the carbon electrode employed in the above-mentioned experiment was made of glassy carbon, graphite, pyrolytic carbon and others are usable for this purpose.
These can be all stably used in nutrient medium.
The change is potential in the process of increase of bacteria can be detected with reliability by using the pairs of solid electrodes, as described above. This contributes an excellent effect toward simplifying the test of sensitivity, as follows:
The apparatus constructed in accordance with the method of the present invention can be expected to be made small-sized and stronglybuilt without the necessity of using a counter electrode of complicated structure such as a hydrogen electrode or a Calomel electrode.
The method of the invention obviates any optical detection, so that it is not subject to the influence of colouration or turbidity of nutrient medium.
The method of the invention needs no special electric current from outside as in the impedance method.
The oxidation-reduction potential between the electrode and the nutrient medium can be read out directly with no interposition and without being influenced by the size and form of the electrodes.
The method of the invention, which detects the rise and fall of the metabolic activity of bacteria, enables even a change of rate of increase of, for example, anaerobic bacteria, which have a slow increase rate, to be reliably determined, and further it is useful for discrimination of inactive bacteria from dead bacteria, and also for judgement of medicinal efficiency by means of bioassay.
Claims (4)
1. A method for measuring the increase of bacteria, which comprises: detecting a change in oxidation-reduction potential of a liquid nutrient medium inoculated with said bacteria, said change being measured as a potential difference between a carbon electrode and a metallic electrode.
2. A method according to claim 1, wherein the metallic electrode is of platinum, titanium, or tungsten.
3. A method according to claim 1 or 2, wherein there is established prior to the measurement a correlation between (i) the time elapsed from the start of the measurement until the potential between the carbon electrode and the metallic electrode markedly changes and (ii) the population of bacteria at that point of time.
4. A method according to claim 1, substantially as described herein with reference to the accompanying drawing.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13891779A JPS5663249A (en) | 1979-10-27 | 1979-10-27 | Measurement of multiplication of bacteria |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| GB2063911A true GB2063911A (en) | 1981-06-10 |
Family
ID=15233156
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB8034498A Withdrawn GB2063911A (en) | 1979-10-27 | 1980-10-27 | Measurement of increase of bacteria |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JPS5663249A (en) |
| DE (1) | DE3040329A1 (en) |
| FR (1) | FR2468648A1 (en) |
| GB (1) | GB2063911A (en) |
| IT (1) | IT1134056B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU586438B2 (en) * | 1984-08-08 | 1989-07-13 | Prutec Ltd. | Method and apparatus for monitoring redox reactions |
| FR2720409A1 (en) * | 1994-05-26 | 1995-12-01 | Food Industry Research Develop | Detecting antimicrobial cpds. e.g. in milk and milk prods. |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1230171A (en) * | 1984-12-21 | 1987-12-08 | Pulp And Paper Research Institute Of Canada | Device for monitoring black liquor oxidation |
| GB8724845D0 (en) * | 1987-10-23 | 1987-11-25 | Metal Box Plc | Detecting microorganisms |
| JP4845594B2 (en) * | 2006-05-29 | 2011-12-28 | 富士通株式会社 | Electrochemical cell and potential application method |
| FR3131636A1 (en) * | 2021-12-30 | 2023-07-07 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | METHOD AND SYSTEM FOR DETECTING AND POSSIBLY IDENTIFYING A MICRO-ORGANISM CONTAINED IN A SAMPLE |
-
1979
- 1979-10-27 JP JP13891779A patent/JPS5663249A/en active Pending
-
1980
- 1980-10-24 FR FR8022837A patent/FR2468648A1/en active Pending
- 1980-10-25 DE DE19803040329 patent/DE3040329A1/en not_active Withdrawn
- 1980-10-27 IT IT25595/80A patent/IT1134056B/en active
- 1980-10-27 GB GB8034498A patent/GB2063911A/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU586438B2 (en) * | 1984-08-08 | 1989-07-13 | Prutec Ltd. | Method and apparatus for monitoring redox reactions |
| FR2720409A1 (en) * | 1994-05-26 | 1995-12-01 | Food Industry Research Develop | Detecting antimicrobial cpds. e.g. in milk and milk prods. |
| GB2289946A (en) * | 1994-05-26 | 1995-12-06 | Food Industry Res & Dev Inst | Determining presence of antimicrobial compounds |
| US5591599A (en) * | 1994-05-26 | 1997-01-07 | Food Industry Research And Development Institute | Method for detecting antimicrobial compounds |
| GB2289946B (en) * | 1994-05-26 | 1998-09-23 | Food Industry Res & Dev Inst | Method |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5663249A (en) | 1981-05-29 |
| FR2468648A1 (en) | 1981-05-08 |
| DE3040329A1 (en) | 1981-05-14 |
| IT8025595A0 (en) | 1980-10-27 |
| IT1134056B (en) | 1986-07-24 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |