CA2312086C - System for analyzing images produced by bacterial reactions - Google Patents
System for analyzing images produced by bacterial reactions Download PDFInfo
- Publication number
- CA2312086C CA2312086C CA002312086A CA2312086A CA2312086C CA 2312086 C CA2312086 C CA 2312086C CA 002312086 A CA002312086 A CA 002312086A CA 2312086 A CA2312086 A CA 2312086A CA 2312086 C CA2312086 C CA 2312086C
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- CA
- Canada
- Prior art keywords
- panel
- support
- reactions
- plate
- images produced
- 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.)
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
Abstract
The system is based on an apparatus (1) forming a closed chamber (2) inside which is arranged a superior and centred digital camera (15) oriented towards a removable support (17) either a tray of wells wherein are situated the liquid cultivating means or a plate on which is placed a solid cultivating means;
the support (17) is arranged between lower lighting means (18) and upper and lateral lighting means (19) and are completed with light diffusers (20 and 21).
The apparatus is provided externally with operation indicators, switches and taps and connections which are intended to the proper operation and control via a central computer. The system captures and interprets the images produced by the reactions which take place in the referred liquid or solid cultivation means, thereby converting said bacterial reactions into values, as well as analyzing the growth of micro-organisms.
the support (17) is arranged between lower lighting means (18) and upper and lateral lighting means (19) and are completed with light diffusers (20 and 21).
The apparatus is provided externally with operation indicators, switches and taps and connections which are intended to the proper operation and control via a central computer. The system captures and interprets the images produced by the reactions which take place in the referred liquid or solid cultivation means, thereby converting said bacterial reactions into values, as well as analyzing the growth of micro-organisms.
Description
SYSTEM FOR ANALYZING IMAGES PRODUCED BY BACTERIAL REACTIONS
DESCRIPTION
OBJECT OF THE INVENTION
The invention refers to a system capable of converting bacterial reaction images into numerical values and analyzing microorganism growt:z by capturing images from a panel or plate.
The object of the invention consists in capturing and interpreting images produced by reactions taking place either in a liquid culture medium contair_ed inside panel receptacles or on the surfGce of a solid culture medium in all-purpose laboratory receptacles, or so-called plates.
BACKGROUND OF THE INVENTION
Liquid culture m_--.diums use support means commonly referred to as panels which are usually made of polystyrene, have a rectangular shape and are fitted with receptacles designed to co:-:~ain various microbiological culture mediums, biochemical substrates and/or antimicrobial agents in liquid form in which metabolic reactions occur, the r~.icrobiological interpretation of which leads to the identification of a microorganism and/or the definition o_ its capacity to be neutralized by one or several antibiotics.
The cultures may also be performed ~~:ith the use of another type of support or plates in the form of a container generally made of polystyrene, having various shapes and containing a single solid cu_~ure medium on which the bacterial reaction ta'.~ces place.
Thus, traditionally, in the case o. the panels, a visual inspection of the reactions occurring the various receptacles i s performed in t:~:e laboratory, and, based on the panels' shape and color, the result probability is established according to general ta'r~les published for this purpose.
DESCRIPTION
OBJECT OF THE INVENTION
The invention refers to a system capable of converting bacterial reaction images into numerical values and analyzing microorganism growt:z by capturing images from a panel or plate.
The object of the invention consists in capturing and interpreting images produced by reactions taking place either in a liquid culture medium contair_ed inside panel receptacles or on the surfGce of a solid culture medium in all-purpose laboratory receptacles, or so-called plates.
BACKGROUND OF THE INVENTION
Liquid culture m_--.diums use support means commonly referred to as panels which are usually made of polystyrene, have a rectangular shape and are fitted with receptacles designed to co:-:~ain various microbiological culture mediums, biochemical substrates and/or antimicrobial agents in liquid form in which metabolic reactions occur, the r~.icrobiological interpretation of which leads to the identification of a microorganism and/or the definition o_ its capacity to be neutralized by one or several antibiotics.
The cultures may also be performed ~~:ith the use of another type of support or plates in the form of a container generally made of polystyrene, having various shapes and containing a single solid cu_~ure medium on which the bacterial reaction ta'.~ces place.
Thus, traditionally, in the case o. the panels, a visual inspection of the reactions occurring the various receptacles i s performed in t:~:e laboratory, and, based on the panels' shape and color, the result probability is established according to general ta'r~les published for this purpose.
Automatic reading systems are presently used which, by measuring the optical density of t'~:~
receptacles, assign a value interpreted as positive cr negative by a computer program. Subsequ°_ntly, anoth=r S computer module establishes the result probability :.~y combining the positive character of the partial reactions.
In the case of the plates, the approximation to the result has traditionally been more rudimentary '-n view of the smaller number of parameters involved, which are based mainly in the amou~:t, size and color of the bacterial colonies.
The advantage of the these automGtic systems which measure optical densities - over the traditi oral visual inspection method lies in t':~at it eliminates t=:~
user's subjectivity in interpreting reactions, sine each reaction is translated into a numerical value.
However, these automatic systems present a seric~.:s problem and dratrrback in that they are unable to interpret both the morphological features and t~~
growth patterns of the bacteria, ~,rhich thus elude t:-:=
result obtaining method of certain important elements, such as the form of bacterial gro:~rth, namely the bas°_ for obtaining rnicrobiological results daring the pest decades.
DESCRIPTION OF THE INVENTION
The proposed system Gras cor_ceived for the purpc=e of solving the above problems, allowing c-.icrobiological reactions to be converted to numerical vGlues which a=a subsequently applied to a diag:~osis software in order to establish the number of r~:i croorga:-:isms prese::_, revealing an infection, to identi=y the microorganis-.s present in the sample or to establish an organis;~.'s antimicrobial susceptibility pattern.
According to the present invention, there is provided a system for analyzing images produced by bacterial reactions, allowing microbiological reactions to be converted into numerical values which are subsequently used by a diagnosis software to determine: the amount of microorganisms present, indicative of an infection; the identification of the microorganism present in the sample; and the infection-causing organism's antimicrobial susceptibility pattern;
wherein the reactions produced by the images which are to be captured and interpreted by means of the system proper occur in liquid culture mediums contained inside panel receptacles or on the surface of a solid culture medium or over a plate, characteristic in that it comprises an apparatus which internally defines a closed hermetic chamber internally fitted with a digital camera mounted on a support, a removable supp~art for the panel or plate concerned, and lighting means and complemented with light diffusers and, whereas externally the apparatus, in correspondence with its front panel, includes a series of luminous indicators, a feeder associated to the corresponding removable support, an opening button acling on the feeder, a contrast switch and a general switch; the rear panel of said apparatus being designed to include a cover providing access to the internal components, as well as outlets, and supplying current to the lighting fixture and to the chamber, and a port for communicating with the central computer, respectively, said rear panel also including the connections to a TDM 436 control module.
The following reactions are identified via the inventive system:
- Bacterial colony size, color, shape and quantity.
- Color development in biochemical substrate receptacles, indicative of the type of bacterial metabolism involved.
- Development of compact shapes or turbidness in antibiotic receptacles that would reflect the microorganism's development ca~~ability in the presence of specific antimicrobial agent concentrations.
receptacles, assign a value interpreted as positive cr negative by a computer program. Subsequ°_ntly, anoth=r S computer module establishes the result probability :.~y combining the positive character of the partial reactions.
In the case of the plates, the approximation to the result has traditionally been more rudimentary '-n view of the smaller number of parameters involved, which are based mainly in the amou~:t, size and color of the bacterial colonies.
The advantage of the these automGtic systems which measure optical densities - over the traditi oral visual inspection method lies in t':~at it eliminates t=:~
user's subjectivity in interpreting reactions, sine each reaction is translated into a numerical value.
However, these automatic systems present a seric~.:s problem and dratrrback in that they are unable to interpret both the morphological features and t~~
growth patterns of the bacteria, ~,rhich thus elude t:-:=
result obtaining method of certain important elements, such as the form of bacterial gro:~rth, namely the bas°_ for obtaining rnicrobiological results daring the pest decades.
DESCRIPTION OF THE INVENTION
The proposed system Gras cor_ceived for the purpc=e of solving the above problems, allowing c-.icrobiological reactions to be converted to numerical vGlues which a=a subsequently applied to a diag:~osis software in order to establish the number of r~:i croorga:-:isms prese::_, revealing an infection, to identi=y the microorganis-.s present in the sample or to establish an organis;~.'s antimicrobial susceptibility pattern.
According to the present invention, there is provided a system for analyzing images produced by bacterial reactions, allowing microbiological reactions to be converted into numerical values which are subsequently used by a diagnosis software to determine: the amount of microorganisms present, indicative of an infection; the identification of the microorganism present in the sample; and the infection-causing organism's antimicrobial susceptibility pattern;
wherein the reactions produced by the images which are to be captured and interpreted by means of the system proper occur in liquid culture mediums contained inside panel receptacles or on the surface of a solid culture medium or over a plate, characteristic in that it comprises an apparatus which internally defines a closed hermetic chamber internally fitted with a digital camera mounted on a support, a removable supp~art for the panel or plate concerned, and lighting means and complemented with light diffusers and, whereas externally the apparatus, in correspondence with its front panel, includes a series of luminous indicators, a feeder associated to the corresponding removable support, an opening button acling on the feeder, a contrast switch and a general switch; the rear panel of said apparatus being designed to include a cover providing access to the internal components, as well as outlets, and supplying current to the lighting fixture and to the chamber, and a port for communicating with the central computer, respectively, said rear panel also including the connections to a TDM 436 control module.
The following reactions are identified via the inventive system:
- Bacterial colony size, color, shape and quantity.
- Color development in biochemical substrate receptacles, indicative of the type of bacterial metabolism involved.
- Development of compact shapes or turbidness in antibiotic receptacles that would reflect the microorganism's development ca~~ability in the presence of specific antimicrobial agent concentrations.
3~
- The formation of a cir~~le around an antibiotic disc, or so-called "inhibiting halo".
The system is thus based on a de~-_ce preferably having a prismatic-rectangular snaps, the inside of which establishes a hermetically clos=_d chamber of diverse shapes and dimensions provided They allow for appropriate focal distance between a digital camera lens and the panel or plate to be analyzed. The digital camera is internally mounted on an upper central support , an internal area ne;~r the lo:~:=r end of the chamber being fitted with a support associated to an inlet/outlet feeder arranged for d=positing the corresponding panels or plate:>, i.e. th= objects to b2 analyzed in each case, characteristic in that the supports are interchangeable ar_d tray be of various types depending on the panels or plates to be processed.
The function of this support installed inside the chamber, as previously stated, is to al_ow the passage of light from the lower part of the object beir_g processed or analyzed, and to ce:~ter the object under the digital camera.
An internal lighting system is also provided, comprising four fluorescent tubes, two of which are d installed along the sides and above the object to be processed and the other two are insta-.led under the object to be processed, so that, depending on the type of image to be processed, the upper, lo~rrer or both fluorescent lights are activated, this being implemented either automatically, throuc:z the junction management software, or manually, by mea::~ of a switch.
Externally, the chamber's front pG~el includes a series of function control LEDs and switc~~es, in addition to the corresponding feeding wechanisms for inserting the panel or plate to be processed.
The chamber's rear panel includes e:~_ternal outlets leading to a central computer, to a TDB: ~--36 ca=d and a to digital camera and lighting syste~:. power supply line; however, depending on the assembly's ope=ational requirements, other outlets may be ins=tilled or said outlets modified.
It must also be noted that the rear panel includes an access cover for internal cleaninc, maintenance, component replacement, calibration, etc., purposes.
The system further includes a mea:Ls of identifying the type of panel and the external data used for complementing the identification tests p°_rformed in the panel, and a means of establishing the p~sition of each receptacle in the panel and calculatir_a the pa:~.el area to be used for the reading.
According to the characteristics ~:~us described, the system, in operation, must fi_-st perform a calibration of the camera by shocuing a c~.mera histogram and its reading values on the screen. of a control personal computer, so that, when a~ indicator lig'-~.ts up to signal the end of the calibration r=tiding, a panel inserted in the internal support of the feeder may be read, said panel's upper surface being divided into three information areas, namely one cc=responding to the type of panel identification Grea, another corresponding to the external information area and a third corresponding to the receptacles aria.
Once the corresponding reading option is activated 5 in the control software, the feeder is closed and an image reading instruction is issued enabling the system-generated data to be interpreted by th' software, which assigns the analyzes bacteria a taxonomic classification and a code as tc whether it .s resistant or not to a given antibiotic or group o.
antibiotics.
DESCRIPTION OF THE DRAWINGS
In order to complement the descri~cion and h_-_--._n toward providing a better understa::ding of t:~e characteristics of the invention according to a preferred practical embodiment therec=, a set o~-_' drawings is attached as an ir_tegral part of t~:~
description wherein the following is =epresented Gs illustrative and non-limiting in charact~=:
Figure 1 shows an external front view of t:e apparatus or chamber which forms a part of the sys~e;,l that is the object of the invention.
Figure 2 shows an external rear vi=w of the sa:~.a apparatus or chamber shown in the preced_:~g figure.
Figure 3 shows a front view sect_on through a vertical plane of the apparatus or chamb== shown in two preceding figures.
Figure ~ shows an external side -riew, also in section through a vertical plane, of the same appara~us or cham~r~er shown in the preceding figure.
Figure 5 shows a general tOp V124: c. a panel w~~h three identification sections correspor_a-zg to: type c-panel, external information, and pos_cion of etch receptacle.
Figure 6 shows a perspective view of a form of embodiment of a receptacle panel support.
Figure 7 shows a further perspective top view of a plate support.
Figure 8 shows a detail of the manner in which a selection of the reading area is perform=d according to the position of each receptacle in the corresponding panel.
Figure 9 shows a plan view of a :.late including several discs impregnated by a selection of antibiotics.
Finally, figure 10 includes a grape corresponding to the calibration histogram.
PREFERRED EMBODIMENT OF THE INVENTION
In the light of the above figures, it is evid=nt that the system of the invention is based on an apparatus fitted with a general housing (1) of a prismatic-rectangular shape which interr_ally defines a closed chamber (2), characteristic in shat its front panel (3) incorporates a series of ext~~nal indicator LEDs (4) providing the following funct_ons: indicator (aa) corresponds to the electric curre_-_t inlet to in the chamber (2) ; indicator (4b) correspc:!ds to the end of the calibration. The three next _-.dicators (~c) correspond to 25%, 50% and 750, respec=ively, of the reading performed; indicator (4d) corr~~ponds to she end of the reading.
Also, said front panel (3) includes a corresponding feeder (5), the functio- of whic:n is described further on, an opening butte-! (6) for the feeder, a contrast switch (7) and a gen==al switch (~), in a manner that said indicators (4) and elements (5 to 8) constitute the means of controlling s_~stem functions and inserting the panel or plate to be read.
The rear panel (9) of apparatus (1) includes a cover (10) providing access to the internal co~aponer.~s and an electric current outlet (11) for the auxiliary lighting systems; an electric current outlet (12) for the digital camera described hereunder; a communication port (13) for connection to the central computer;
connections (14) to a TDNI 436 module containing t:~:~
calculating program for converting the image's calorimetric signals into numeric values, sa~-d connections (14) corresponding to blue, green and rid colors which provide the signals corresponding to the percentages of said colors or colorimetr'_c signals.
Inside chamber (2) is fitted said digital camera (15), mounted on an upper support (16), she camera (1~) being arranged in a central position any in lines wi=h the center of a support ( 17 ) on w:~ich try obj ect to be analyzed is placed, namely the correspo~:ding plate or panel. Support (17) is interchangeable and may vary in shape, depending on the feeder (5) ar.~ the type of panel or plate to be supported. In any case, sa-d support (17) is part of the feeding dev_ce (5) itsel=, and constitutes the means allowing for t_~? insertion of the supports to be processed, i.e. the panels and plates, since pressing the opening batten (6) involves sliding the feeder (5) and thus the support (17) guided over rails and activated on its outward r..ovement by a-:y conventional system, from a simple pressure spring to a pneumatic mechanism or similar device.
Inside chamber (2), in addition tc said digital camera (15) and support (17), are inc,_uded light-~:g means in the form of a pair of fluorescent lamps (lo) located on a lo:.rer positi on and a pair of fluoresce:-:t lamps (19) located at an intermediate lateral heigr~, said fluorescent lights (18) and (19) bs,_~:g complemented with respective light diffusers (20) and (21), so that the former lie under the object to be processed, namely under the support (17), whereas the latter lie above it, characteristic in that the latter are complemented with a protective plate (19') forming an inclined metal screen preventing the radiations to interfere with the readings of the digital camera (15).
The light diffusers (20) and (21) are preferably mane of white crystal and are fitted for providing homogeneous light distribution, in the first case on the object to be processed and in t_~e second case through the transparent polystyrene panel or plate receptacles.
Depending on the type of image to be processes, the upper fluorescent lights (19) or the loc,:er fluorescent lights (18), or both simui~aneously, a=a activated either automatically, throug'~ the softwara managing the various functions, or manually, through a contrast switch (7) located on the front panel (3) .
Figure 6 shows a support (17') fo= a receptacla panel, which support (17') is fitted 4:ith holes (22) for the panel's receptacles and recesses (23) and (2"-_) for illuminating the panels identification area a-:d the external information area, said support (17') also including kingpins (25) for attac:~.:~ent to t~;e corresponding feeder.
Figure 7 shows a plate support (17") fitted wv'h kingpins for attachment to the feeder, i-: addition tc a hole (26) for the lower lighting of t~:e upper plate, and attachment flanges (27) for the platy itself.
Operation of the apparatus varies depending on cahether the purpose is to analyze the i~::age of a pa-:~l with its biochemical reactions and antimicrob~:a1 susceptibility patterns occurring in different sectic:~s of the same panel, or the image of a plate with a colony growth and/or inhibiting halos.
In any case, prior to the reading session corresponding to the same type of product, panel c.
plate, the apparatus must be calibrated after selecting the support (17) to be used, which in turn is d~pende.-_~
on the panel or plate to be processed.
Thus, once the support (17) is installed, which Gs previously stated must be shaped as shoran in Figure =, reference (17'), or shaped as shown in Figure 7, reference (17"), or otherwise shaped depending on t'ce type of panel or plate, prior to inserting a panel cr plate, in order to allow the light to pass free~:y through holes (22) or (26) through the support, t~?
'calibrate' option is executed from the control software residing in a personal computer a-d communicated with the apparatus through sa_d communications port (13). The camera then shows t:~e histogram and reading values on the control personal computer screen, as shown in the graph in Figure 1~, while indicator (4b) is activated to ir_dicate the end of the calibration reading.
If the calibration is within stable ranges, t~
process comes to an end; otherwise, chamber (c) adjustments are necessary.
The histogram provided by the system, as shown _n Figure 10, must have its end p°_aks and valley as near as possible to the abscise axis.
The reading is performed as folio=.as In the case of panels which, as previously state, are polystyrene supports fitted wits a number c~
receptacles containing biochemical su'nstrates used _;r identifying a bacteria and others co::taining gro~~,=h concentrations of antimicrobial agents used establishing a bacteria's capacity to withstand t=a attack with the lowest possible antibiot_c concentration-, the process starts by putting a readi::g algorithm to work which divides the panel's surface (28), shown in Figure 5, into t:~ree sectior_s corresponding to references (29), (30) and (31), with reference (29) corresponding to the Hype of panel 5 identification area, reference (30) to the external information area and reference (31) to t'ne receptacles area.
In the first case, and as previous_y stated, area (29) identifies the type of panel bei:-:g used (three 10 different types are available, according to microorganism, group of microorganisms or samples of microorganism sources). Area (29) is diz-ided into four rectangles, although other geometrical shapes are acceptable, printed preferably in the upper left-hand section of panel (28) (for example, if the first and third receptacles in area (29) are fu~-1, this wound identify an enterobacteria panel). Evidently, information on the type of panel is essential at the time of interpreting the response of the bacteria when faced with certain antibiotics.
Concerning area (30), foreseen for identifying external information used in corm-ementing the identification tests included in the panel, the area comprises six squares, although other geometrical shapes are allowed, in w'nich the user marks a point covering at least 90% of the surface if =he bacteria in question have reacted positively tJ a sp=cific laboratory technique (for example, a po rt cover-_r_g tre first square of the upper portion woulc indicat= that the microorganism has reacted to the cul=ure process at 18:00 hours as a result of an oxidase test).
Concerning the receptacles zone or area (31), t~is area establishes the position of each re~°_ptacle t:rithin the panel and calculates the panel's reading area, generating four values for each receptacle.
As shown in Figure 8, wherein =eference (32) corresponds to the digital camera (1) focus, the receptacles (33) are cylindrical, the lower part or bottom (33') thereof being conical in s?:ape; also, the selection of the area to be considered in respect to the vertical of the digital camera fixed-.y installed in the upper part of the apparatus is essential for detecting possible bacterial growth. I- said figure, references (34) correspond to the liquid culture medium and references (35) to the bacterial g-owth detection area, which are different in each case d=pending on t'::e location of the corresponding receptacle (33) -n respect to the vertical of the digital camera (3%) focus.
Out of the four values generated b-.~ the system -n the course of processing a panel, the fi=~ two are us.d for detecting bacterial metabolism. calorimetr-c reactions, corresponding to color cc~e and color saturation, whereas the third and fou_~.h values a=a used for detecting bacterial growth, cc=responding ~o the growth area, and the amount of light passing though each receptacle.
The values obtained for each recep ~acle (33 ) a=a then tabulated by the control softwar= in order ~o identify the microorganism causing the infection a~:d the bast antibiotics against said infec=ion, in the_r lower concentration.
Fi gure 9 s:zows a plate ( 35 ) in .he form o. a circular polystyrene support, which may acqui=a different shapes and sizes, containing G solid cultu=a medium on the surface of wzich is extend?d t'::e microorganism suspension presumably causing t:~e infection.
After inoculating the whole sur=ace with tie bacterial suspension, a selection o. antibiotics-impregnated discs (37) is deposited thereon, the antibiotics contained in the discs th=_n dissolving in and around the inoculated culture medium to achie-re lower antibiotic concentrations in points further acaay S from the edge of the disc. In said Figure 9, the zoo:?
under reference (38) is the perimete-= reconstruction zone resulting from the superposition of the 'inhibiting halos' from the two discs (37).
The capacity of the microorganisms to grow arou_-?d an antibiotics disc, to a greater or lesser extent or none at a11, determines their classification Gs sensitive or resistant to said antibiotic and thus establishes the antibiotic's usefulness as an agent for fighting against the infection.
The microorganism forms a circul a-r zone aro~~:~:d each disc, referred to above as an _=~hibiting halo', which is measured by the apparatus.
Thus, once the plate (36) is _nserted in the chamber (2) and the order is issued to process t.~~e image, a reading algorithm is activated which divides the surface into as many sections as discs are deposited on the medium. It then verif--es that no halos are superposed, which is a phenomeno-_ resulting from two discs located near each other gene=acing a halo of a diameter such that both overlap, so that, in tie event of a superposition, the system reconstructs t:~:?
most probable halo for each disc, name--y the previou=ly mentioned zone (38) .
The average diameter of each ir:hibiting halo is then calculated on the basis of direc~ measurement of the diameters as such, or some other parameter lead_ng to this information. Three measuremer_~s are perforr-..d on each halo in order to calculate the average diameter, although a different number of measuremen~s could likewise be implemented.
On completing the measurements, the system associates each measurement to a disc (37), according to its location on the plate (36), starting from th?
one on the upper portion and advancing clockwise.
The values obtained for each disc (37) are then tabulated by the control software in order to determine the antibiotics that are most convenient for fighting against the microorganism causing the infection.
Obviously, in addition to the panel and plate readings according to the previous d?scription, a colony recount must be conducted, this naturally consisting in counting the number of bacterial groups that have managed to grow on the surface of a solid medium; according to the object o. the invention, said 1S colony recount starts with a techniaue consisting in "sowing" a bacterial suspension on the surface of a medium contained on a plate, incubating it at 37°C and establishing the number of groups (color:ies) appeari r_a after an 18- to 1~-hour incubation: period.
The system and the means associated thereto perform a color assessment of the base r.edium on which the suspension was inoculated, the numeric values ranging from 0 to 256.
In this respect, a colony is considered to be the smallest number of adjacent pixels snowing a 2Go difference in color above or below the base nedium value.
Once the surface of a colony is established, said surface is extrapolated to all the areas presenting a 20o difference and a calculation is made as to ho~f~ many times the surface of a colony is contained in t:~:e overall surface; the value obtained is then tabul ated, this operation being performed by the control software associated to the system in order to decide whether or not the number of colonies is indicative of the presence of an infectious process.
- The formation of a cir~~le around an antibiotic disc, or so-called "inhibiting halo".
The system is thus based on a de~-_ce preferably having a prismatic-rectangular snaps, the inside of which establishes a hermetically clos=_d chamber of diverse shapes and dimensions provided They allow for appropriate focal distance between a digital camera lens and the panel or plate to be analyzed. The digital camera is internally mounted on an upper central support , an internal area ne;~r the lo:~:=r end of the chamber being fitted with a support associated to an inlet/outlet feeder arranged for d=positing the corresponding panels or plate:>, i.e. th= objects to b2 analyzed in each case, characteristic in that the supports are interchangeable ar_d tray be of various types depending on the panels or plates to be processed.
The function of this support installed inside the chamber, as previously stated, is to al_ow the passage of light from the lower part of the object beir_g processed or analyzed, and to ce:~ter the object under the digital camera.
An internal lighting system is also provided, comprising four fluorescent tubes, two of which are d installed along the sides and above the object to be processed and the other two are insta-.led under the object to be processed, so that, depending on the type of image to be processed, the upper, lo~rrer or both fluorescent lights are activated, this being implemented either automatically, throuc:z the junction management software, or manually, by mea::~ of a switch.
Externally, the chamber's front pG~el includes a series of function control LEDs and switc~~es, in addition to the corresponding feeding wechanisms for inserting the panel or plate to be processed.
The chamber's rear panel includes e:~_ternal outlets leading to a central computer, to a TDB: ~--36 ca=d and a to digital camera and lighting syste~:. power supply line; however, depending on the assembly's ope=ational requirements, other outlets may be ins=tilled or said outlets modified.
It must also be noted that the rear panel includes an access cover for internal cleaninc, maintenance, component replacement, calibration, etc., purposes.
The system further includes a mea:Ls of identifying the type of panel and the external data used for complementing the identification tests p°_rformed in the panel, and a means of establishing the p~sition of each receptacle in the panel and calculatir_a the pa:~.el area to be used for the reading.
According to the characteristics ~:~us described, the system, in operation, must fi_-st perform a calibration of the camera by shocuing a c~.mera histogram and its reading values on the screen. of a control personal computer, so that, when a~ indicator lig'-~.ts up to signal the end of the calibration r=tiding, a panel inserted in the internal support of the feeder may be read, said panel's upper surface being divided into three information areas, namely one cc=responding to the type of panel identification Grea, another corresponding to the external information area and a third corresponding to the receptacles aria.
Once the corresponding reading option is activated 5 in the control software, the feeder is closed and an image reading instruction is issued enabling the system-generated data to be interpreted by th' software, which assigns the analyzes bacteria a taxonomic classification and a code as tc whether it .s resistant or not to a given antibiotic or group o.
antibiotics.
DESCRIPTION OF THE DRAWINGS
In order to complement the descri~cion and h_-_--._n toward providing a better understa::ding of t:~e characteristics of the invention according to a preferred practical embodiment therec=, a set o~-_' drawings is attached as an ir_tegral part of t~:~
description wherein the following is =epresented Gs illustrative and non-limiting in charact~=:
Figure 1 shows an external front view of t:e apparatus or chamber which forms a part of the sys~e;,l that is the object of the invention.
Figure 2 shows an external rear vi=w of the sa:~.a apparatus or chamber shown in the preced_:~g figure.
Figure 3 shows a front view sect_on through a vertical plane of the apparatus or chamb== shown in two preceding figures.
Figure ~ shows an external side -riew, also in section through a vertical plane, of the same appara~us or cham~r~er shown in the preceding figure.
Figure 5 shows a general tOp V124: c. a panel w~~h three identification sections correspor_a-zg to: type c-panel, external information, and pos_cion of etch receptacle.
Figure 6 shows a perspective view of a form of embodiment of a receptacle panel support.
Figure 7 shows a further perspective top view of a plate support.
Figure 8 shows a detail of the manner in which a selection of the reading area is perform=d according to the position of each receptacle in the corresponding panel.
Figure 9 shows a plan view of a :.late including several discs impregnated by a selection of antibiotics.
Finally, figure 10 includes a grape corresponding to the calibration histogram.
PREFERRED EMBODIMENT OF THE INVENTION
In the light of the above figures, it is evid=nt that the system of the invention is based on an apparatus fitted with a general housing (1) of a prismatic-rectangular shape which interr_ally defines a closed chamber (2), characteristic in shat its front panel (3) incorporates a series of ext~~nal indicator LEDs (4) providing the following funct_ons: indicator (aa) corresponds to the electric curre_-_t inlet to in the chamber (2) ; indicator (4b) correspc:!ds to the end of the calibration. The three next _-.dicators (~c) correspond to 25%, 50% and 750, respec=ively, of the reading performed; indicator (4d) corr~~ponds to she end of the reading.
Also, said front panel (3) includes a corresponding feeder (5), the functio- of whic:n is described further on, an opening butte-! (6) for the feeder, a contrast switch (7) and a gen==al switch (~), in a manner that said indicators (4) and elements (5 to 8) constitute the means of controlling s_~stem functions and inserting the panel or plate to be read.
The rear panel (9) of apparatus (1) includes a cover (10) providing access to the internal co~aponer.~s and an electric current outlet (11) for the auxiliary lighting systems; an electric current outlet (12) for the digital camera described hereunder; a communication port (13) for connection to the central computer;
connections (14) to a TDNI 436 module containing t:~:~
calculating program for converting the image's calorimetric signals into numeric values, sa~-d connections (14) corresponding to blue, green and rid colors which provide the signals corresponding to the percentages of said colors or colorimetr'_c signals.
Inside chamber (2) is fitted said digital camera (15), mounted on an upper support (16), she camera (1~) being arranged in a central position any in lines wi=h the center of a support ( 17 ) on w:~ich try obj ect to be analyzed is placed, namely the correspo~:ding plate or panel. Support (17) is interchangeable and may vary in shape, depending on the feeder (5) ar.~ the type of panel or plate to be supported. In any case, sa-d support (17) is part of the feeding dev_ce (5) itsel=, and constitutes the means allowing for t_~? insertion of the supports to be processed, i.e. the panels and plates, since pressing the opening batten (6) involves sliding the feeder (5) and thus the support (17) guided over rails and activated on its outward r..ovement by a-:y conventional system, from a simple pressure spring to a pneumatic mechanism or similar device.
Inside chamber (2), in addition tc said digital camera (15) and support (17), are inc,_uded light-~:g means in the form of a pair of fluorescent lamps (lo) located on a lo:.rer positi on and a pair of fluoresce:-:t lamps (19) located at an intermediate lateral heigr~, said fluorescent lights (18) and (19) bs,_~:g complemented with respective light diffusers (20) and (21), so that the former lie under the object to be processed, namely under the support (17), whereas the latter lie above it, characteristic in that the latter are complemented with a protective plate (19') forming an inclined metal screen preventing the radiations to interfere with the readings of the digital camera (15).
The light diffusers (20) and (21) are preferably mane of white crystal and are fitted for providing homogeneous light distribution, in the first case on the object to be processed and in t_~e second case through the transparent polystyrene panel or plate receptacles.
Depending on the type of image to be processes, the upper fluorescent lights (19) or the loc,:er fluorescent lights (18), or both simui~aneously, a=a activated either automatically, throug'~ the softwara managing the various functions, or manually, through a contrast switch (7) located on the front panel (3) .
Figure 6 shows a support (17') fo= a receptacla panel, which support (17') is fitted 4:ith holes (22) for the panel's receptacles and recesses (23) and (2"-_) for illuminating the panels identification area a-:d the external information area, said support (17') also including kingpins (25) for attac:~.:~ent to t~;e corresponding feeder.
Figure 7 shows a plate support (17") fitted wv'h kingpins for attachment to the feeder, i-: addition tc a hole (26) for the lower lighting of t~:e upper plate, and attachment flanges (27) for the platy itself.
Operation of the apparatus varies depending on cahether the purpose is to analyze the i~::age of a pa-:~l with its biochemical reactions and antimicrob~:a1 susceptibility patterns occurring in different sectic:~s of the same panel, or the image of a plate with a colony growth and/or inhibiting halos.
In any case, prior to the reading session corresponding to the same type of product, panel c.
plate, the apparatus must be calibrated after selecting the support (17) to be used, which in turn is d~pende.-_~
on the panel or plate to be processed.
Thus, once the support (17) is installed, which Gs previously stated must be shaped as shoran in Figure =, reference (17'), or shaped as shown in Figure 7, reference (17"), or otherwise shaped depending on t'ce type of panel or plate, prior to inserting a panel cr plate, in order to allow the light to pass free~:y through holes (22) or (26) through the support, t~?
'calibrate' option is executed from the control software residing in a personal computer a-d communicated with the apparatus through sa_d communications port (13). The camera then shows t:~e histogram and reading values on the control personal computer screen, as shown in the graph in Figure 1~, while indicator (4b) is activated to ir_dicate the end of the calibration reading.
If the calibration is within stable ranges, t~
process comes to an end; otherwise, chamber (c) adjustments are necessary.
The histogram provided by the system, as shown _n Figure 10, must have its end p°_aks and valley as near as possible to the abscise axis.
The reading is performed as folio=.as In the case of panels which, as previously state, are polystyrene supports fitted wits a number c~
receptacles containing biochemical su'nstrates used _;r identifying a bacteria and others co::taining gro~~,=h concentrations of antimicrobial agents used establishing a bacteria's capacity to withstand t=a attack with the lowest possible antibiot_c concentration-, the process starts by putting a readi::g algorithm to work which divides the panel's surface (28), shown in Figure 5, into t:~ree sectior_s corresponding to references (29), (30) and (31), with reference (29) corresponding to the Hype of panel 5 identification area, reference (30) to the external information area and reference (31) to t'ne receptacles area.
In the first case, and as previous_y stated, area (29) identifies the type of panel bei:-:g used (three 10 different types are available, according to microorganism, group of microorganisms or samples of microorganism sources). Area (29) is diz-ided into four rectangles, although other geometrical shapes are acceptable, printed preferably in the upper left-hand section of panel (28) (for example, if the first and third receptacles in area (29) are fu~-1, this wound identify an enterobacteria panel). Evidently, information on the type of panel is essential at the time of interpreting the response of the bacteria when faced with certain antibiotics.
Concerning area (30), foreseen for identifying external information used in corm-ementing the identification tests included in the panel, the area comprises six squares, although other geometrical shapes are allowed, in w'nich the user marks a point covering at least 90% of the surface if =he bacteria in question have reacted positively tJ a sp=cific laboratory technique (for example, a po rt cover-_r_g tre first square of the upper portion woulc indicat= that the microorganism has reacted to the cul=ure process at 18:00 hours as a result of an oxidase test).
Concerning the receptacles zone or area (31), t~is area establishes the position of each re~°_ptacle t:rithin the panel and calculates the panel's reading area, generating four values for each receptacle.
As shown in Figure 8, wherein =eference (32) corresponds to the digital camera (1) focus, the receptacles (33) are cylindrical, the lower part or bottom (33') thereof being conical in s?:ape; also, the selection of the area to be considered in respect to the vertical of the digital camera fixed-.y installed in the upper part of the apparatus is essential for detecting possible bacterial growth. I- said figure, references (34) correspond to the liquid culture medium and references (35) to the bacterial g-owth detection area, which are different in each case d=pending on t'::e location of the corresponding receptacle (33) -n respect to the vertical of the digital camera (3%) focus.
Out of the four values generated b-.~ the system -n the course of processing a panel, the fi=~ two are us.d for detecting bacterial metabolism. calorimetr-c reactions, corresponding to color cc~e and color saturation, whereas the third and fou_~.h values a=a used for detecting bacterial growth, cc=responding ~o the growth area, and the amount of light passing though each receptacle.
The values obtained for each recep ~acle (33 ) a=a then tabulated by the control softwar= in order ~o identify the microorganism causing the infection a~:d the bast antibiotics against said infec=ion, in the_r lower concentration.
Fi gure 9 s:zows a plate ( 35 ) in .he form o. a circular polystyrene support, which may acqui=a different shapes and sizes, containing G solid cultu=a medium on the surface of wzich is extend?d t'::e microorganism suspension presumably causing t:~e infection.
After inoculating the whole sur=ace with tie bacterial suspension, a selection o. antibiotics-impregnated discs (37) is deposited thereon, the antibiotics contained in the discs th=_n dissolving in and around the inoculated culture medium to achie-re lower antibiotic concentrations in points further acaay S from the edge of the disc. In said Figure 9, the zoo:?
under reference (38) is the perimete-= reconstruction zone resulting from the superposition of the 'inhibiting halos' from the two discs (37).
The capacity of the microorganisms to grow arou_-?d an antibiotics disc, to a greater or lesser extent or none at a11, determines their classification Gs sensitive or resistant to said antibiotic and thus establishes the antibiotic's usefulness as an agent for fighting against the infection.
The microorganism forms a circul a-r zone aro~~:~:d each disc, referred to above as an _=~hibiting halo', which is measured by the apparatus.
Thus, once the plate (36) is _nserted in the chamber (2) and the order is issued to process t.~~e image, a reading algorithm is activated which divides the surface into as many sections as discs are deposited on the medium. It then verif--es that no halos are superposed, which is a phenomeno-_ resulting from two discs located near each other gene=acing a halo of a diameter such that both overlap, so that, in tie event of a superposition, the system reconstructs t:~:?
most probable halo for each disc, name--y the previou=ly mentioned zone (38) .
The average diameter of each ir:hibiting halo is then calculated on the basis of direc~ measurement of the diameters as such, or some other parameter lead_ng to this information. Three measuremer_~s are perforr-..d on each halo in order to calculate the average diameter, although a different number of measuremen~s could likewise be implemented.
On completing the measurements, the system associates each measurement to a disc (37), according to its location on the plate (36), starting from th?
one on the upper portion and advancing clockwise.
The values obtained for each disc (37) are then tabulated by the control software in order to determine the antibiotics that are most convenient for fighting against the microorganism causing the infection.
Obviously, in addition to the panel and plate readings according to the previous d?scription, a colony recount must be conducted, this naturally consisting in counting the number of bacterial groups that have managed to grow on the surface of a solid medium; according to the object o. the invention, said 1S colony recount starts with a techniaue consisting in "sowing" a bacterial suspension on the surface of a medium contained on a plate, incubating it at 37°C and establishing the number of groups (color:ies) appeari r_a after an 18- to 1~-hour incubation: period.
The system and the means associated thereto perform a color assessment of the base r.edium on which the suspension was inoculated, the numeric values ranging from 0 to 256.
In this respect, a colony is considered to be the smallest number of adjacent pixels snowing a 2Go difference in color above or below the base nedium value.
Once the surface of a colony is established, said surface is extrapolated to all the areas presenting a 20o difference and a calculation is made as to ho~f~ many times the surface of a colony is contained in t:~:e overall surface; the value obtained is then tabul ated, this operation being performed by the control software associated to the system in order to decide whether or not the number of colonies is indicative of the presence of an infectious process.
Claims (6)
1. A system for analyzing images produced by bacterial reactions, allowing microbiological reactions to be converted into numerical values which are subsequently used by a diagnosis software to determine:
the amount of microorganisms present, indicative of an infection; the identification of the microorganism present in the sample; and the infection-causing organism s antimicrobial susceptibility pattern;
wherein the reactions produced by the images which are to be captured and interpreted by means of the system proper occur in liquid culture mediums contained inside panel receptacles or on the surface of a solid culture medium or over a plate, characteristic in that it comprises an apparatus (1) which internally defines a closed hermetic chamber (2) internally fitted with a digital camera (15) mounted on a support (16), a removable support (17) for the panel or plate concerned, and lighting means (18) and (19) complemented with light diffusers (20) and (21), whereas externally the apparatus (1), in correspondence with its front panel (3), includes a series of luminous indicators (4), a feeder (5) associated to the corresponding removable support (17), an opening button (6) acting on the feeder (5), a contrast switch (7) and a general switch (8); the rear panel (9) of said apparatus (1) being designed to include a cover (10) providing access to the internal components, as well as outlets (11), (12) and (1.3) supplying current to the lighting fixture and to the chamber, and a port for communicating with the central computer, respectively, said rear panel (9) also including the connections (14) to a TDM 436 control module.
the amount of microorganisms present, indicative of an infection; the identification of the microorganism present in the sample; and the infection-causing organism s antimicrobial susceptibility pattern;
wherein the reactions produced by the images which are to be captured and interpreted by means of the system proper occur in liquid culture mediums contained inside panel receptacles or on the surface of a solid culture medium or over a plate, characteristic in that it comprises an apparatus (1) which internally defines a closed hermetic chamber (2) internally fitted with a digital camera (15) mounted on a support (16), a removable support (17) for the panel or plate concerned, and lighting means (18) and (19) complemented with light diffusers (20) and (21), whereas externally the apparatus (1), in correspondence with its front panel (3), includes a series of luminous indicators (4), a feeder (5) associated to the corresponding removable support (17), an opening button (6) acting on the feeder (5), a contrast switch (7) and a general switch (8); the rear panel (9) of said apparatus (1) being designed to include a cover (10) providing access to the internal components, as well as outlets (11), (12) and (1.3) supplying current to the lighting fixture and to the chamber, and a port for communicating with the central computer, respectively, said rear panel (9) also including the connections (14) to a TDM 436 control module.
2. A system for analyzing images produced by bacterial reactions, according to claim 1, characteristic in that the support (17) is mounted in a coupling/uncoupling relationship with the feeder (5), the latter being guided in its inward and outward movements to allow for the corresponding receptacle panel (28) or plate (36) to be inserted.
3. A system for analyzing images produced by bacterial reactions, according to claim 1, characteristic in that the support (17') for the receptacle panels (28) is shaped according to the shape of the panels, said support being fitted with specific lateral recesses (23) and (24) and with kingpins (25) for attachment to the feeder (5), and also holes (22) for the receptacles (33) of the corresponding panel (18).
4. A system for analyzing images produced by bacterial reactions, according to claim 1, characteristic in that the support (17") for the plate (36) is fittted with kingpins (25) for attachment to the feeder (5) and a hole (26) for the disc that constitutes the plate (36), in addition to flanges (27) for attaching the plate.
5. A system for analyzing images produced by bacterial reactions, according to claim 1, characteristic in that, on the upper surface of panel (28), three areas (29), (30) and (31) are established, the first one for identifying the type of panel, the second for identifying the external information and the third for identifying the receptacle, with areas (29) and (30) corresponding to recesses (23) and (24) in the support (17).
6. A system for analyzing images produced by bacterial reactions, according to claim 1, characteristic in that the support (17) fitted inside. the chamber (2) of apparatus (1) is located above the lighting means wich constitute the fluorescent lights (18) and below the lighting means which constitute the fluorescent lights (19), the former being installed in the lower portion, whereas the lighting means or fluorescent light tubes (19) are located along the sides at an intermediate height, both being complemented by light diffusers (20) and (21), while the fluorescent light tubes (19) are complemented with metal plates (19') which interrupt the trajectory of the light beam between the upper digital camera (15) and the panel over support (17).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ESP9702500 | 1997-12-02 | ||
| ES009702500A ES2137879B1 (en) | 1997-12-02 | 1997-12-02 | ANALYZING SYSTEM OF IMAGES PRODUCED BY BACTERIAL REACTIONS. |
| PCT/ES1998/000303 WO1999028436A1 (en) | 1997-12-02 | 1998-11-11 | System for analyzing images produced by bacterial reactions |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2312086A1 CA2312086A1 (en) | 1999-06-10 |
| CA2312086C true CA2312086C (en) | 2005-08-16 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002312086A Expired - Fee Related CA2312086C (en) | 1997-12-02 | 1998-11-11 | System for analyzing images produced by bacterial reactions |
Country Status (10)
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|---|---|
| US (1) | US6642953B1 (en) |
| EP (1) | EP1074610B1 (en) |
| JP (1) | JP2001525162A (en) |
| AT (1) | ATE321840T1 (en) |
| CA (1) | CA2312086C (en) |
| DE (1) | DE69834051T2 (en) |
| DK (1) | DK1074610T3 (en) |
| ES (2) | ES2137879B1 (en) |
| PT (1) | PT1074610E (en) |
| WO (1) | WO1999028436A1 (en) |
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-
1998
- 1998-11-11 AT AT98952748T patent/ATE321840T1/en not_active IP Right Cessation
- 1998-11-11 DK DK98952748T patent/DK1074610T3/en active
- 1998-11-11 CA CA002312086A patent/CA2312086C/en not_active Expired - Fee Related
- 1998-11-11 DE DE69834051T patent/DE69834051T2/en not_active Expired - Fee Related
- 1998-11-11 PT PT98952748T patent/PT1074610E/en unknown
- 1998-11-11 EP EP98952748A patent/EP1074610B1/en not_active Expired - Lifetime
- 1998-11-11 US US09/554,512 patent/US6642953B1/en not_active Expired - Fee Related
- 1998-11-11 JP JP2000523314A patent/JP2001525162A/en not_active Withdrawn
- 1998-11-11 WO PCT/ES1998/000303 patent/WO1999028436A1/en active IP Right Grant
-
2004
- 2004-09-17 ES ES200402223A patent/ES2270666B1/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| ATE321840T1 (en) | 2006-04-15 |
| ES2137879B1 (en) | 2000-08-16 |
| CA2312086A1 (en) | 1999-06-10 |
| DK1074610T3 (en) | 2006-06-19 |
| EP1074610A1 (en) | 2001-02-07 |
| US6642953B1 (en) | 2003-11-04 |
| DE69834051D1 (en) | 2006-05-18 |
| ES2270666B1 (en) | 2007-12-01 |
| JP2001525162A (en) | 2001-12-11 |
| PT1074610E (en) | 2006-07-31 |
| DE69834051T2 (en) | 2006-10-26 |
| ES2270666A1 (en) | 2007-04-01 |
| ES2137879A1 (en) | 1999-12-16 |
| EP1074610B1 (en) | 2006-03-29 |
| WO1999028436A1 (en) | 1999-06-10 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |