GB2178847A - Testing for the presence of living organisms at the surface of an object - Google Patents

Testing for the presence of living organisms at the surface of an object Download PDF

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Publication number
GB2178847A
GB2178847A GB08519814A GB8519814A GB2178847A GB 2178847 A GB2178847 A GB 2178847A GB 08519814 A GB08519814 A GB 08519814A GB 8519814 A GB8519814 A GB 8519814A GB 2178847 A GB2178847 A GB 2178847A
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Prior art keywords
detection device
luminescence
living organisms
photo
atp
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GB08519814A
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GB8519814D0 (en
Inventor
Douglas Irish
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Philips Electronics UK Ltd
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Philips Electronic and Associated Industries Ltd
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Priority to GB08519814A priority Critical patent/GB2178847A/en
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Publication of GB2178847A publication Critical patent/GB2178847A/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76Chemiluminescence; Bioluminescence
    • G01N21/763Bioluminescence
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06Quantitative determination
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Plasma & Fusion (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

A method of determining the quantity of living organisms at the surface of an object involves applying a bioluminescent agent, for example firefly luciferin-luciferase enzyme, onto the surface, conveniently by spraying. NRB nucleotide releasing reagent is then sprayed onto the surface wetted with the enzyme solution. As the ATP-bearing cells of any micro organisms present at the surface of the sample are disrupted by the releasing agent, the ATP is released into an environment of firefly enzyme whereby bioluminescence is emitted in the immediate vicinity of the cell from which the ATP was released. The luminescence is then monitored by apparatus which has a display device which may be calibrated to indicate the level of the emitted luminescence, or where a particular organism is to be detected directly in quantity of organism per unit area.

Description

SPECIFICATION Testing for the presence of living organisms at the surface of an object The invention relates to a method of determining the quantity of living organisms at the surface of an object, the living organisms having cells bearing a biochemical substance which is capable of reaction with a specific enzyme to produce bioluminescence comprising the steps of applying to the surface an agentforthe release of the biochemical substance from the cells, providing a bioluminescent agent for reaction with the released biochemical substance to produce bioluminescence and detecting the luminescence produced. The invention further relates to appar atusforcarrying outthemethod.
It has been known for several years that all living organisms contain adenosinetriphosphate (ATP), and that hydrolytic reaction of ATP with certain enzymes results in the production of light, although the mechanism of the reaction is not necessarily fully understood. The amount of light, referred to as bioluminescence, emitted is very small, and it has been the practice, in laboratory experiments with the reaction, to determine the light emission by use of a photometer.
The ATP is contained in cells within the organisms, and can be released from the cells by the application of a reagent capable of attacking the cell membrance. it has now been realised thatthe reaction can be adapted to provide a simple, rapid and essentially non-destructive testforthe contamination by micro-organisms of the surface of objects such as pieces of textile fabric.
ATP is, in fact, oniy one of a group of biochemical substances, each of which is capable of reaction with a specific enzyme to produce bioluminescence, and although in the following specification reference will be made for the sake of simplicity primarily to ATP, it should be understood that the term is to be construed as extending to any memberofthe aforementioned group ora derivative thereof.
U.K. Patent Appl ication No. 211 6709A describes and claims a method oftestforthe presence of living organisms having ATP-bearing cells atthe surface of an object, comprising applying to the surface an agent for the release of ATP from the cells, providing a bioluminescent agentfor reaction with released ATP to produce bioluminescence, and observing the luminescence produced.
That application suggests that the luminescence can be seen bytheeye or recorded byacamera with the aid of an image intensifier. It also suggests that by providing the image intensifier with a video camera and recording system, and provided thatthe reagents applied do not inhibitthe growth ofthe micro organisms, the technique may provide a method for observing and recording the developing extent of contamination as a function of time.However, image intensifiers are normally operated to give a constant output brightness once a threshold image brightness has been exceeded and consequently a determination of thequantityoforganismsatagiven point is not feasible by viewing the bioluminescence through the image intensifier or by recording the image by means of a camera.
It is an object of the invention to enable a quantitative determination ofthe presence of living organisms atthesurface of an object.
The invention provides a method of determining the quantity of living organisms at the surface of an object as set forth in the opening paragraph characterised in that the luminescence is monitored bya detection device which produces an electrical output which is dependent on the inten sity of the luminescencefalling on the detection device, that the electrical output is converted to a measure of the quantity of the living organisms present, and that the converted output is arranged to drive a display device.
The invention further provides apparatus arranged to carry out the method set forth in the preceding paragraph, the apparatus comprising a detection device for receiving the luminescence and converting the received luminescence to an electrical signal whose magnitude is dependent on the intensity of the received luminescence, means for converting the electrical signal to a signal which is representative of the quantity of living organisms present in a given area, and means for displaying the converted signal.
The method and apparatus set forth in the two preceding paragraphs have the advantage over the method disclosed in U.K. PatentApplication No.
2116707Athat a quantitativedetermination of the living organisms present at the surface of an object can be conveniently made. Although that application makes reference to semi-quantitative determination by estimation of the luminescence seen through an image intensifierthis is only feasible over a limited range of intensities since the image intensifierwill either saturate or have its gain reduced by an automatic gain control arrangement once a threshold value has been reached. Another semi-quantatitive approach suggested by the cited application is to repeatedly photograph the image intensifier output over a period of time and thus record the change in area of contamination.This is a time consuming task and will only indicate whetherthe area of contamination is changing and will not give a reliable indication ofthe degree of contamination ata particular point.
The detection device may comprise a photo-diode or a photo-transistor. Alternatively the detection device may comprise a photomultipliertube.
These alternatives allow a comparatively inexpensive monitoring apparatus to be constructed and in particularwhen a photo-diode or photo-transistor is used a portable battery operated apparatus may be constructed. There are other devices which may be used as the detection device, for examplethe electrical output of the automatic gain control circuit of an image inten sifiercould be used providedthatthelowerdetection limit, which is determined bythethreshold illumination at which the automatic gain circuit starts to operate, is sufficiently lowforthe required sensitivity of detection.
In order to concentrate the maximum illumination on the detection device a lens or mirror may be provided forfocussing the received luminescence onthe detection device. Thiswill increase the detection sensitivity of the apparatus.
The display means maycomprisea digital voltmeter. This can be a comparatively compact and inexpensive item enabling a digital display of the quantity of organisms present An embodiment of the invention will now be described, by way of example, with reference to the accompanying drawings, in which Figure 1 shows in partial cross section apparatus for carrying outthe method of determining the quantity of living organisms at the surface of an object; and Figure2shows a circuit diagram of the apparatus of Figure 1. A method according to the inventin of determining the quantity of living organismsatthesurfaceofan object involves applying a bioluminescent agent, for example Firefly luciferin-luciferase enzyme, onto the surface, conveniently by spraying.No significant reaction with ATP from surface contamination occurs atthis stage because theATP is predominantly enclosed within cell membrances. Commercially available firefly enzyme may itself be slightlycontaminated with ATP however and there may be a slight bioluminescent background from reaction ofthe enzyme with such traces of ATP, and such background luminescence should be discounted in considering that revealed afterthe next subsequent step.
NRB nucleotide releasing reagent is then sprayed onto the surfacewetted with the enzyme solution. As the ATP-bearing cells of any micro organisms present at the surface of the sample are disrupted by the releasing agent, the ATP is released into an environment offirefly enzyme whereby bioluminescence is emitted in the immediate vicinity of the cell from which theATPwas released.
For instruction on the preparation of the Firefly luciferin-luciferase enzyme reference should be made to the aforementioned patent application.
The luminscence is then monitored by apparatus which is described hereinafter to determine the level of the emitted bioluminescence. The apparatus has a display device which may be calibrated to indicate the level of the emitted luminescence, or where a particular organism isto be detected directly in quantity of organism per unit area.
Figure 1 shows in partial cross-section apparatus for carrying outthe method described. An ashown in Figure 1 the apparatus comprises a body 1 ofany arbitrary cross-section, for example circular or rectangular, the body 1 having an opening 2 through which light can pass to a lens3 mounted within the body 1. The lens 3 is arranged to focus the light passing through the opening 2 onto a detector 4, which may be a photo-diode. The detector is connected in an electrical circuit such as that shown in Figure 2. A display device 5 is mounted in the body 1 to provide an indication of the quantity of organisms present in an area corresponding to the area of the opening 2.To perform the methodtheATPand NRB nucleotide releasing reagent are sprayed on the surface 6 of an object to be investigated and the organisms 7then luminesce. The body 1 is then placed over the luminescing organisms so that the emitted radiation passes through the opening 2 and is focussed on the detector4. A reading isthen produced on the display device 5. It is, of course, important to exclude ambient light fro the detector 4 and thus the opening 2 must be placed in contact with the surface ofthe object.
Figure 2 shows the detector 4 in the form of a photodiode which is connected to the input of an operational amplifier Al. The parallel arrangement of a resistor R2 and a capacitor C1 is connected between the input and output of the amplifier Al. The series arrangement of three resistors R3, R4 and R5 is connected between the output of the amplifier Al and the terminal 11. A selection switch S1 has three selection contacts which are connected to the output of the amplifier Al, the junction of resistors R3 and R4 and the junction of resistors R4 and R5, respectively. The pole of the switch S1 is connected to the input of a digital voltmeter DVM the display of which forms the display device 5.
Thus in this em bodiment the photodiode4forms the detection device, the operational amplifier and digital voltmeter form the conversion means, and the display on the digital voltmeter forms the means for displaying the converted signal. Clearly the conversion means may comprise further signal processing circuitry to linearisethe response, to determine the measurement time, and to reduce the effects of noise. Such signal processing circuitrywould be well known to those skilled in the art.
A photo-diode or photo-transistor form convenient detection devices since they enable a low voltage powersupplyto be used enabling a compact battery operated unit to be constructed.
However if greater sensitivity is required they would be replaced buy a photomultipliertube or an image intensifier as discussed hereinbefore. The lens 3 may be omitted ifthedetector4issuffici- ently sensitive. Since the purpose of the lens 3 isto converge the emitted luminescence on the detector it could be replaced by other optical elements such as a mirror.
The method may be used forthe monitoring of microbiological contamination of a textile fabric, for example mildew on woollen cloth, orfor contamination of other surfaces, for example in hospital operating threatres, and can be carried out simplywithouthandling oftheobjects.

Claims (12)

1. A method of determining the quantity of living organisms at the surface of an object, the living organisms having cells bearing a biochemical substance which is capable of reaction with a specific enzyme to produce bioluminescence comprising the steps of applying the surface an agentforthe release of the biochemical substance from the cells, providing a bioluminescent agent for reaction with the released biochemical substance to produce bioluminescence and detecting the luminescence produced characterised in that the luminance is monitored buy a detection device which produces an electrical outputwhich is dependent on the intensity of the luminescence falling on the detection device, thatthe electrical output is converted to a measure of the quantity of the living organisms present in a given area, and that the converted output is arranged to drive a display device.
2. A method as claimed in Claim 1, characterised in that the detection device comprises a photo-diode our a photo-transistor.
3. A method as claimed in Claim 1, characterised in that the detection device comprises a photomultipliertube.
4. A method as claimed in any preceding claim, characterised in that the luminescence is focussed on the detection device by a lens.
5. A method of determining the quantity of living organisms at the surface of an object sub- stantially as described herein with reference to the accompanying drawings.
6. Apparatus arranged to carry out the method of Claim 1, the apparatus comprising a detection device for receiving the luminescence and converting the received luminescenceto an electrical signal whose magnitude is dependent on the intensity of the received luminescence, means for converting the electrical signal to a signal which is representative of the quantity of living organisms present in a given area, and means for displaying the converted signal.
7. Apparatus as claimed in Claim 6, in which the detection devicecomprises a photo-diodeora photo-transistor.
8. Apparatus as claimed in Claim 6, in which the detection device comprises a photomultiplier tube.
9. Apparatus as claimed in any of Claims 6to 8, comprising means for focussing the received luminescence on the detection device.
10. Apparatus as claimed in Claim 9, in which the focussing means comprises a lens.
11. Apparatus as claimed in any of Claims 6 to 10, in which the display means comprises a digital voltmeter.
12. Apparatus arranged to carry outthe method of Claim 1, the apparatus being substantially as described herein with reference to the accompanying drawings.
GB08519814A 1985-08-07 1985-08-07 Testing for the presence of living organisms at the surface of an object Withdrawn GB2178847A (en)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3737649A1 (en) * 1987-11-06 1989-05-24 Inst Zellforschung Und Biolumi Method for determining the luminescence of cell cultures, and device for carrying out the method
GB2218803A (en) * 1988-05-20 1989-11-22 Gerald Whitlock Light photon detecting apparatus
WO1990004775A1 (en) * 1988-10-21 1990-05-03 Biotrace Ltd. Luminescence or fluorescence monitoring apparatus and method
WO1991009300A1 (en) * 1989-12-08 1991-06-27 Image Research Limited Improvements in and relating to light transfer systems and improved cell investigation techniques arising therefrom
GB2281966A (en) * 1993-09-07 1995-03-22 Biotrace Ltd Luminometer
WO1996007755A2 (en) * 1994-09-09 1996-03-14 Lachema A.S Method of performing confirmatory tests by fluorochrome substrates in indicators of fecal contamination or potentially pathogenic bacteria
GB2294115A (en) * 1994-10-11 1996-04-17 Whitlock Hughes Ltd Measuring surface contamination by organisms
WO2001011344A1 (en) * 1999-08-09 2001-02-15 Medical Packaging Corporation Hygiene monitoring apparatus, system and uses thereof
GB2358124A (en) * 2000-01-14 2001-07-18 Chemisphere Uk Ltd Method of cleaning and monitoring in ware washing machines
WO2005059162A2 (en) * 2003-12-16 2005-06-30 Kimberly-Clark Worldwide, Inc. Microbial detection and quantification
US7300770B2 (en) 2004-12-16 2007-11-27 Kimberly-Clark Worldwide, Inc. Detection of microbe contamination on elastomeric articles
US7399608B2 (en) 2003-12-16 2008-07-15 Kimberly-Clark Worldwide, Inc. Microbial detection and quantification
US7531319B2 (en) 2006-08-31 2009-05-12 Kimberly-Clark Worldwide, Inc. Array for rapid detection of a microorganism
US7727513B2 (en) 2005-12-15 2010-06-01 Kimberly-Clark Worldwide, Inc. Method for screening for bacterial conjunctivitis
US7763442B2 (en) 2006-08-31 2010-07-27 Kimberly-Clark Worldwide, Inc. Method for detecting candida on skin

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2001434A (en) * 1977-07-19 1979-01-31 Tarkkanen V Measurement of somatic cells in milk
EP0025350A2 (en) * 1979-09-05 1981-03-18 Dynatech Ag Apparatus for detecting luminescent reactions
GB1601031A (en) * 1977-01-31 1981-10-21 Plakas C J Method for detection of bacterial concentration by luminescence
GB2073885A (en) * 1980-04-15 1981-10-21 Whitlock G D Method of and apparatus for detecting the presence of live organisms in substances

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1601031A (en) * 1977-01-31 1981-10-21 Plakas C J Method for detection of bacterial concentration by luminescence
GB2001434A (en) * 1977-07-19 1979-01-31 Tarkkanen V Measurement of somatic cells in milk
EP0025350A2 (en) * 1979-09-05 1981-03-18 Dynatech Ag Apparatus for detecting luminescent reactions
GB2073885A (en) * 1980-04-15 1981-10-21 Whitlock G D Method of and apparatus for detecting the presence of live organisms in substances

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3737649A1 (en) * 1987-11-06 1989-05-24 Inst Zellforschung Und Biolumi Method for determining the luminescence of cell cultures, and device for carrying out the method
GB2218803B (en) * 1988-05-20 1992-09-09 Gerald Whitlock Light photon detecting apparatus
GB2218803A (en) * 1988-05-20 1989-11-22 Gerald Whitlock Light photon detecting apparatus
US5108175A (en) * 1988-05-20 1992-04-28 Gerald Whitlock Light photon detecting apparatus
WO1990004775A1 (en) * 1988-10-21 1990-05-03 Biotrace Ltd. Luminescence or fluorescence monitoring apparatus and method
JPH04502403A (en) * 1988-10-21 1992-05-07 バイオトレイス リミテッド Luminescence or Fluorescence Monitoring Apparatus and Method
WO1991009300A1 (en) * 1989-12-08 1991-06-27 Image Research Limited Improvements in and relating to light transfer systems and improved cell investigation techniques arising therefrom
AU651801B2 (en) * 1989-12-08 1994-08-04 Cambridge Imaging Limited Improvements in and relating to light transfer systems and improved cell investigation techniques arising therefrom
GB2281966A (en) * 1993-09-07 1995-03-22 Biotrace Ltd Luminometer
GB2281966B (en) * 1993-09-07 1997-06-04 Biotrace Ltd Bio-luminescence monitoring method
WO1996007755A2 (en) * 1994-09-09 1996-03-14 Lachema A.S Method of performing confirmatory tests by fluorochrome substrates in indicators of fecal contamination or potentially pathogenic bacteria
WO1996007755A3 (en) * 1994-09-09 1996-07-25 Lachema As Method of performing confirmatory tests by fluorochrome substrates in indicators of fecal contamination or potentially pathogenic bacteria
GB2294115A (en) * 1994-10-11 1996-04-17 Whitlock Hughes Ltd Measuring surface contamination by organisms
WO2001011344A1 (en) * 1999-08-09 2001-02-15 Medical Packaging Corporation Hygiene monitoring apparatus, system and uses thereof
GB2358124A (en) * 2000-01-14 2001-07-18 Chemisphere Uk Ltd Method of cleaning and monitoring in ware washing machines
GB2358124B (en) * 2000-01-14 2003-08-20 Chemisphere Uk Ltd Method of cleaning and monitoring in ware washing machines
US8338128B2 (en) 2003-12-16 2012-12-25 Kimberly-Clark Worldwide, Inc. Microbial detection and quantification
WO2005059162A2 (en) * 2003-12-16 2005-06-30 Kimberly-Clark Worldwide, Inc. Microbial detection and quantification
WO2005059162A3 (en) * 2003-12-16 2005-09-15 Kimberly Clark Co Microbial detection and quantification
US7282349B2 (en) 2003-12-16 2007-10-16 Kimberly-Clark Worldwide, Inc. Solvatochromatic bacterial detection
US7399608B2 (en) 2003-12-16 2008-07-15 Kimberly-Clark Worldwide, Inc. Microbial detection and quantification
US7687245B2 (en) 2003-12-16 2010-03-30 Kimberly-Clark Worldwide, Inc. Microbial detection and quantification
US7300770B2 (en) 2004-12-16 2007-11-27 Kimberly-Clark Worldwide, Inc. Detection of microbe contamination on elastomeric articles
US7727513B2 (en) 2005-12-15 2010-06-01 Kimberly-Clark Worldwide, Inc. Method for screening for bacterial conjunctivitis
US7531319B2 (en) 2006-08-31 2009-05-12 Kimberly-Clark Worldwide, Inc. Array for rapid detection of a microorganism
US7763442B2 (en) 2006-08-31 2010-07-27 Kimberly-Clark Worldwide, Inc. Method for detecting candida on skin
US8361742B2 (en) 2006-08-31 2013-01-29 Kimberly-Clark Worldwide, Inc. Method for detecting Candida on skin
US8617874B2 (en) 2006-08-31 2013-12-31 Kimberly-Clark Worldwide, Inc. Array for rapid detection of a microorganism

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