Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
• • 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
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/04—Flat or tray type, drawers
• • 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
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/14—Scaffolds; Matrices
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
  • G06V10/00—Arrangements for image or video recognition or understanding
  • G06V10/10—Image acquisition
  • G06V10/12—Details of acquisition arrangements; Constructional details thereof
  • G06V10/14—Optical characteristics of the device performing the acquisition or on the illumination arrangements
  • G06V10/141—Control of illumination
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
  • G06V20/00—Scenes; Scene-specific elements
  • G06V20/60—Type of objects
  • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
  • G06V20/693—Acquisition

Definitions

• a sample suspected of containing "a" microorganism can be interpreted to mean that the sample can include “one or more” microorganisms.
• Figure 2 is a top view, partially in section, of one embodiment of a culture device with a contrast layer according to the present disclosure.
• Figure 4b is an enlarged view of a portion of the culture device of FIG. 4a.
• Figure 5a is a top view of the culture device of FIG. 3 at a third point in time.
• the present disclosure is generally directed to methods and articles for detecting microorganisms in a sample.
• the articles and methods employ substantially optically-transmissive culture devices.
• the low haze-high clarity devices provide the high contrast needed to distinguish a colony of microorganisms from the materials of the culture device and provide the high spatial resolution to distinguish two microbial colonies that are located very close to one another.
• the methods employ the observation of the diminution or disappearance of one or more abiogenic gas bubbles in a culture device as an early indication (e.g., before the appearance of a visible colony) of the presence of a microorganism in a culture device.
• substantially optically -transmissive refers to an optical path in which the optical haze, as measured by ASTM Method 1003, is less than or equal to about 95% and the optical clarity, as measured by ASTM Method 1003, is greater than or equal to about 10%.
• An "abiogenic gas bubble”, as used herein, refers to an optically-detectable gas bubble that is produced by means other than biological activity. "Optically-detectable” is used in the broadest sense and includes visual detection by a human as well as detection by machine vision.
• FIG. 1 illustrates an embodiment of a culture device 1 10 in accordance with the present disclosure.
• the culture device 1 10 comprises a base 1 12 and a cover sheet 122.
• the culture device 1 10 includes an outermost first major surface 104 and an outermost second major surface 106.
• the culture device 1 10, when inoculated with a liquid sample, is configured to form a highly transmissive optical path extending from the first major surface to the second major surface.
• Optional adhesive layer 1 14 can comprise a variety of adhesives (e.g., pressure- sensitive adhesives) known in the art.
• suitable adhesives include copolymer silicone adhesives (e.g., the silicone adhesives described in U.S. Patent No. 6,703, 120, for example, which is incorporated herein by reference in its entirety) and acrylate adhesives (e.g., the acrylate adhesives described in U.S. Patent No. 4,565,783, for example, which is incorporated herein by reference in its entirety).
• the adhesive layer 1 14 can be applied to the base member 1 12 and/or the cover sheet 122 using processes known in the art (e.g., knife- coating, extrusion, laminative transfer from a release liner, etc.).
• a template e.g., a weighted ring (not shown) may be applied temporarily to the outside of cover sheet, after closing, to confine the sample to a specific region while the cold-water-soluble powder forms a gel.
• the portion of the culture device inoculated with a sample generally delineates the growth area 126 of the device.
• Table 1 Composition of an exemplary culture medium.
• the culture medium can comprise a buffer. Suitable buffers include phosphate buffers.
• the carbonate buffer is a sodium carbonate buffer.
• the phosphate buffer is a potassium phosphate buffer.
• the culture medium can comprise more than one buffering agent (e.g., potassium phosphate and sodium acetate).
• the phosphate buffer can be about 22 mM.
• the quantity of each component of the dry culture medium is at least partially determined by at least two factors: (1) the concentration of that component in the culture medium solution, and (2) the amount of the solution coated onto a given surface area of the culture device (the coating weight). Suitable coating weights may range from about
• the contrast layer 230 may comprise a reflective layer, such as metal, metal foil, a metalized polymeric film, or a mirror, for example.
• the contrast layer 230 may comprise a specularly-reflective layer, such as the specularly- reflective film.
• An example of a suitable specularly-reflective layer is a Vikuiti Enhanced Specular Reflective (ESR) Film (part number 98044027500) obtained from 3M Company in St. Paul, MN.
• ESR Vikuiti Enhanced Specular Reflective
• the contrast layer 230 may be coupled (e.g., adhesively coupled) to the culture device 210.
• the contrast layer 230 may be a composition that is coated onto the base member 212 of the culture device 210.
• the present disclosure provides methods for detecting bacteria in a sample.
• the method comprises providing a liquid sample and a culture device comprising a base member, a cover layer, and adhesive layer coupled to the base member and or cover sheet, and a dry cold water-soluble gelling agent disposed on the adhesive layer, the device configured to form a highly transmissive optical path.
• the method further comprises observing an indication of growth.
• the indication of growth may be observed by a person visually.
• the indication of growth may be observed using an imaging device.
• the imaging device may be display or print an image of the growth area such that a person can visually observe and analyze the displayed or printed image.
• FIG. 6 shows one embodiment of detecting the presence or absence of a microorganism in a sample according to the present disclosure.
• the embodiment includes providing a culture device 610 (shown in side view) configured to form a highly-transmissible optical path extending from an outermost first major surface 604 to an outermost second major surface 606.
• the culture device 610 includes a base member 612, an optional adhesive layer 614 coupled to the base member 612, a cover sheet 622, and a hydrogel 617 (e.g., a hydrated gelling layer) disposed between the adhesive layer 614 and the cover sheet 622.
• the hydrogel 617 is hydrated with a liquid sample.
• observing an indication of microbial growth further comprises providing an optical filter 650 and positioning the optical filter 650 between the light source 670 and the culture device 610.
• the culture device 610 can be illuminated with selected wavelengths of light (e.g., red wavelengths, blue wavelengths, ultraviolet wavelengths). This configuration can be particularly advantageous when the color of a microbial colony 660 is only slightly different from the color of one or more components of the culture device 610.
• observing an indication of microbial growth can comprise observing the culture device with the observer facing the second major surface of the culture device.
• detecting the presence or absence of a microorganism can comprise detecting the scattering, absorbance or transmittance of light by a colony of microorganisms and/or by an indicator reagent.
• observing an indication of microbial growth further comprises providing a contrast layer 630 and, prior to observing the culture device 610, positioning the contrast layer 630 proximate the second major surface 606 of the culture device 610.
• the contrast layer 630 substantially reflects or absorbs the photons from the light source 670 and thereby increasing the contrast between the microbial colony 660 and one or more of the components of the culture device 610.
• Suitable contrast layers 630 of these embodiments include any of the contrast layers described herein.
• the contrast layer 630 can be positioned such that it overlaps all or any portion of the growth area (not shown) of the culture device 610.
• observing an indication of microbial growth further comprises, prior to observing the culture device, positioning a second contrast layer proximate the second major surface of the culture device, illuminating the culture device from a light source positioned facing the first major surface of the culture device, and observing an indication of microbial growth.
• These embodiments may be particularly advantageous in detecting two distinct microorganisms (e.g., distinct species; distinct genera; and distinct groups such as coliforms, for example).
• these embodiments may provide for the detection and differentiation of microorganisms.
• these embodiments may provide for the confirmation of a particular species, genus, or group of microorganisms.
• the first contrast layer may provide the observation of a presumptive indication (e.g., C0 2 production from the metabolism of lactose by a coliform microorganism) of the presence of a particular microorganism and the second contrast layer may provide the observation of a confirmatory indication (e.g., hydrolysis of a chromogenic enzyme substrate for the ⁇ -galactosidase enzyme found in coliform microorganisms) of the presence of the microorganism.
• observing an indication of growth comprises observing the growth area from an observation position facing the first major surface of the culture device.
• observing an indication of microbial growth further comprises detecting an observable change in an indicator reagent (e.g., a pH indicator reagent or a chromogenic or fluorogenic enzyme substrate).
• the indicator reagent may change to a derivative (e.g., a protonated indicator reagent, a hydrolysed indicator reagent) wherein the change in the indicator reagent (e.g., a color change or fluorescence change) provides an observable bright spot, dark spot, or color change in and/or adjacent the microbial colony 660.
• Suitable indicator reagents include, for example fluorescent or fluorogenic molecules and bioluminescent compounds.
• observing an indication of microbial growth comprises positioning an observer facing the first major surface of a culture device and positioning a light source facing the second major surface of the culture device, as shown in FIG. 7.
• the culture device 10 is illuminated by photons from a light source 770.
• the photons can generally pass through the optically-transmissive culture device 710.
• a microorganism present in the original sample can form a microbial colony 760 in the gelling layer 716 of the culture device 710.
• Photons from the light source 770 can strike the microbial colony 760 where they can be reflected (shown as photon "A", which is reflected toward observer 790), transmitted, or absorbed, causing an observable bright spot, dark spot or colored spot that contrasts with the brightness and/or color of the components of the culture device 710 (e.g., the cover layer, the gelling layer, the adhesive layer, and/or the base member).
• detecting the presence or absence of a microorganism can comprise detecting the scattering, absorbance or transmittance of light by a colony of microorganisms and/or by an indicator reagent. Detecting the Presence or Absence of a Microorganism by Observing an Abiogenic Gas Bubble:
• the present disclosure provides methods for detecting the presence or absence of a microorganism by observing the presence or size of an abiogenic gas bubble.
• the methods comprise providing a sample and a culture device comprising a base member, a cover layer, and a hydrogel disposed there between.
• the hydrogel includes a plurality of abiogenic gas bubbles distributed therein.
• the hydrogel defines a growth area in the culture device.
• the methods further comprise inoculating the growth area of the device with the sample, incubating the device for a period of time, illuminating the culture device with a light source, and detecting the presence or absence of a microorganism in the culture device by detecting the diminution or absence of at least one abiogenic gas bubble in the culture device at a first point in time.
• microorganism or group of microorganisms e.g., antibiotic -resistant microorganisms.
• the nutrients and/or selective agents to favor the growth of a particular organism or group of organisms are known to a person of ordinary skill in the art.
• the nutrient or selective agent should be selected such that it does not substantially interfere with the formation and/or observation of abiogenic gas bubbles in the hydrogel.
• the hydrogel is in uniform contact with the base member and cover layer of the culture device throughout the growth area of the device (i.e., the hydrogel is "sandwiched" between the base member and the cover layer).
• the culture device can be a thin film culture device such as the devices disclosed in U.S. Patent No. 4,565,783.
• the culture device can be a highly optically-transmissive culture device according to the present disclosure.
• the number and spatial distribution of abiogenic gas bubbles can facilitate the detection of a microorganism in the culture device.
• the culture device can be observed for an indication of the presence or absence of a microorganism.
• the presence of a microorganism can be indicated by a discontinuity of a plurality of abiogenic gas bubbles in the culture device at a first point in time. That is, the culture device can be observed for the presence of a plurality of abiogenic gas bubbles distributed in the growth area.
• a discontinuity can be observed when, within a region of the growth area, one or more of the abiogenic gas bubbles are substantially smaller than the typical abiogenic gas bubbles in the growth area and/or there is an area in which one or more abiogenic gas bubbles are absent.
• the abiogenic gas bubbles may diminish in size or disappear by the metabolism of gasses (oxygen, carbon dioxide, and/or nitrogen) by a microorganism or by the microbial production of surfactants that disperse one or more abiogenic gas bubbles.
• gasses oxygen, carbon dioxide, and/or nitrogen
• surfactants that disperse one or more abiogenic gas bubbles.
• the presence of a discontinuity of abiogenic gas bubbles in the culture device can be more apparent when the culture device is observed (or imaged) at two or more time points (e.g., before substantial microbial growth has occurred and after microbial growth has occurred) and the observations at each point in time are compared.
• observing an indication of growth further comprises positioning a contrast layer on the major surface of the culture device opposite the major surface that the observer (e.g., the operator or an imaging device) is facing.
• detecting the presence or absence of a microorganism can comprise detecting the scattering, absorbance or transmittance of light (e.g., scattering, absorbance, or transmittance of light by a microbial colony).
• detecting the presence or absence of a microorganism can comprise enumerating microorganisms.
• Non-limiting examples of suitable imaging systems include PETRIFILM Plate Reader (PPR), available from 3M Company (St. Paul, MN), the PETRIFILM Plate Reader (PPR), available from 3M Company (St. Paul, MN), the PETRIFILM Plate Reader (PPR), available from 3M Company (St. Paul, MN), the PETRIFILM Plate Reader (PPR), available from 3M Company (St. Paul, MN), the PETRIFILM Plate Reader (PPR), available from 3M Company (St. Paul, MN), the
• obtaining an image comprises obtaining a wavelength-biased image.
• the imaging system can include a bias filter that biases the light collected by the imaging device.
• Filter elements are known in the art and include both "cut-off filters (i.e., filters that allow the passage of light wavelengths either above or below a certain specified wavelength) and "band-pass" filters (i.e., filters that allow the passage of light wavelengths between certain specified upper and lower limits).
• a bias filter can be positioned between the illumination source and the culture device. Alternatively or additionally, a bias filter can be positioned between the culture device and the imaging device.
• a processor 894 controls the operation of imaging device 892. Also shown in FIG. 8 is optional display 876, which can receive an image from the processor 894 for visual review by an operator. In operation, processor 894 controls imaging device 892 to illuminate the culture device 882 and obtain an image. Processor 894 receives image data representing the scanned image from imaging device 892. In some embodiments, processor 894 can select an image, from multiple images, for analysis and/or display. Processor 894 analyzes at least one image of culture device 882 and may produce an analytical result, such as a count of colonies of microorganisms or a determination of the presence or absence of microorganisms in a sample. The analytical result (e.g., a qualitative or quantitative result) can be displayed on display 876, stored in optional data storage memory 898, or retrieved by a host computer (not shown) via optional communication port 895
• Analyzing the image of the culture device can comprise using a system to detect color and/or varying shades of a color (e.g., red, green, blue, gray) in an image.
• Suitable image analysis systems include the image analysis systems described in, for example, U.S. Patent Nos. 5,448,652; 6,243,486; and 6, 153,400; each of which is incorporated herein by reference in its entirety.
• the image may be a composite image made by combining two or more images collected while illuminating the culture device with two or more different sources of relatively narrow- spectrum visible light (e.g., red, green, or blue light).
• the image may be an image collected while illuminating the culture device with a source of relatively narrow- spectrum visible light (e.g., green light).
• certain wavelengths of the image can be selected for displaying or printing an image and/or image analysis.
• Embodiment 1 is a method for detecting the presence or absence of a microorganism in a sample, comprising:
• Embodiment 3 is the method according to embodiment 1 or embodiment 2, wherein illuminating the growth area comprises illuminating the growth area with the light source positioned facing the first major surface of the culture device.
• Embodiment 5 is the method according to embodiment 1 or embodiment 2, wherein illuminating the growth area comprises illuminating the growth area with the light source positioned facing the second major surface of the culture device.
• Embodiment 6 is the method according to embodiment 5, wherein observing an indication of growth comprises observing the growth area from a position facing the first major surface of the culture device.
• the first contrast layer prior to detecting the presence or absence of a microorganism, positioning the first contrast layer proximate the second major surface of the culture device.
• Embodiment 9 is the method of embodiment 8, further comprising:
• Embodiment 10 is the method of any one of embodiments 7 through 9, wherein at least one contrast layer substantially reflects the light.
• Embodiment 12 is the method of any one of embodiments 7 through 9, wherein at least one contrast layer substantially absorbs selected wavelengths of light.
• Embodiment 14 is the method of any one of the preceding embodiments, wherein detecting the presence or absence of a microorganism comprises detecting the scattering, absorbance or transmittance of light.
• Embodiment 17 is the method of any one of the preceding embodiments, wherein detecting the presence or absence of a microorganism comprises detecting a fluorescent signal.
• Embodiment 18 is a method for detecting the presence or absence of a microorganism in a sample, comprising:
• the culture device comprises an outermost first major surface and an outermost second major surface
• hydrogel defines a growth area
• detecting the presence or absence of a microorganism comprises observing an indication of growth
• Embodiment 19 is the method of embodiment 18; wherein providing the culture device comprises providing a thin film culture device that includes a dry, cold water-soluble gelling agent and wherein the method further comprises hydrating the gelling agent with an aqueous liquid.
• Embodiment 22 is the method of any one of embodiments 18 through 21 , further comprising:
• Embodiment 23 is the method of any one of embodiments 18 through 22, wherein illuminating the growth area comprises illuminating the growth area with the light source positioned facing the first major surface of the culture device.
• Embodiment 24 is the method of any one of embodiments 18 through 23, wherein observing an indication of growth comprises observing the growth area from an observation position facing the first major surface of the culture device.
• Embodiment 25 is the method of any one of embodiments 18 through 22, wherein illuminating the growth area comprises illuminating the growth area with the light source positioned facing the second major surface of the culture device.
• Embodiment 27 is the method of any one of embodiments 18 through 22 wherein, overlapping at least a portion of the growth area of the culture device, the second major surface of the culture device comprises a contrast layer.
• Embodiment 28 is the method of embodiment 22, further comprising:
• Embodiment 29 is the method of embodiment 28, further comprising:
• Embodiment 30 is the method of any one of embodiments 27 through 29, wherein at least one contrast layer substantially reflects the light.
• Embodiment 31 is the method of any one of embodiments 27 through 29, wherein at least one contrast layer is a specularly reflective layer
• Embodiment 32 is the method of any one of embodiments 27 through 29, wherein at least one contrast layer substantially absorbs selected wavelengths of light.
• Embodiment 34 is the method of any one of the preceding embodiments, wherein detecting the presence or absence of a microorganism comprises detecting the scattering, absorbance or transmittance of light.
• Embodiment 36 is the method of any one of the preceding embodiments, wherein detecting the presence or absence of a microorganism comprises enumerating microorganisms.
• Embodiment 37 is the method of any one of the preceding embodiments, wherein detecting the presence or absence of a microorganism comprises detecting and differentiating two or more types of microorganisms.
• Embodiment 38 is the method of any one of the preceding embodiments, wherein detecting the presence or absence of a microorganism further comprises:
• observing an indication of growth comprises displaying, printing, or analyzing the image of the growth area.
• Embodiment 39 is a device for detecting microorganisms, comprising:
• the device is substantially optically transmissible when the gelling agent is hydrated with a clear aqueous liquid.
• Embodiment 42 is the device of embodiment 41 , further comprising a nutrient medium disposed on the first or second adhesive layer.
• Embodiment 43 is the device of any one of embodiments 40 through 42, further comprising an indicator reagent.
• Embodiment 45 is the device of any one of embodiments 40 through 44, wherein the optical haze of the culture device after hydration of the device with a clear aqueous liquid is ⁇
• Embodiment 46 is the device of any one of embodiments 40 through 45, wherein the optical clarity of the culture device after hydration of the device with a clear aqueous liquid is > 10% when measured according to ASTM 1003.
• a pressure-sensitive adhesive coated polyolefin film having a silicone pressure-sensitive adhesive (3MTM Advanced Polyolefin Diagnostic Tape Catalog # 9795R available from 3M Company, St. Paul, MN) was used to prepare a powder coated base member for a thin film culture device.
• the tape had a 2-mil thick clear polyolefin backing and a 2-mil thick layer of silicone pressure-sensitive adhesive, and a white release liner.
• a powder nutrient composition was prepared by thoroughly mixing a powder containing 14.3% (all percentages are weight %) pancreatic digest of casein, 25.5% meat peptone, 9.5% yeast extract, 43.4% dextrose, 6% pepticase, l%ferric ammonium citrate, 0.3% calcium chloride, and sufficient sodium carbonate to adjust the pH to about 7.
• a gelling powder composition was prepared by mixing in a 1 : 1 weight ratio xanthan gum powder and locust bean gum powder (both available from Spectrum Chemical Mfg. Corp (Gardena, CA)). Sufficient silica (Cab-O-Sil M5 available from Cabot Corp. (Bilerica, MA) was added to enhance flow and prevent clumping.
• the amount is less than about 0.5 weight %.
• the nutrient composition was mixed with the gelling powder composition in a 1 :4 weight ratio.
• a base member was prepared by sprinkling an excess of the mixed powder composition onto the adhesive coated side of the tape. Excess powder was removed by tilting the sheet and tapping lightly by hand.
• a powder gelling composition was prepared by mixing 500 grams of xanthan gum powder (NF Grade Xanthan Gum, obtained from Spectrum Chemical Manufacturing Corp., Gardena, CA) and 500 grams of locust bean gum (FCC Grade Locust Bean gum obtained from Spectrum Chemical Manufacturing Corp., Gardena, CA) with about 2.9 grams of silica (Cab- O-Sil M5).
• a cover sheet was prepared by sprinkling the powder composition onto a clear adhesive coated tape (3MTM Advanced Polyolefin Diagnostic Tape Catalog # 9795R) and removing the excess powder by tilting the sheet and tapping lightly by hand.
• Rectangular plate devices measuring 3 inches by 3.75 inches were cut from the coated sheet to form a base member, and from the coated film to form a cover film.
• a thin film culture device was assembled by adhering a strip of double coated pressure-sensitive adhesive tape on one end the coated base plate and adhering one end of the cover sheet to it so that the coated surfaces faced each other and the tape functioned as a hinge to hold the cover sheet to the base member Fig. 1.
• a thin film culture device was prepared according to the procedure of Example 1 except that the nutrient composition for the base member was prepared by thoroughly mixing of 22.8 parts of pancreatic digest of casein, 15.9 parts of yeast extract, 45.5 parts of sodium pyruvate, 4.1 parts of dextrose, 9.0 parts of dibasic potassium phosphate, and 2.8 grams of monobasic potassium phosphate.
• a powder gelling composition was prepared by mixing guar gum (Ml 50 guar MEYPROGAT gum, Meyhall Chemical AG) with a sufficient amount of silica (Cab-O-Sil M5 silica) to prevent clumping and enhance flow (less than about .5%)
• a cover sheet was prepared according to the procedure of Example 1 except that the powder gelling agent composition was prepared by mixing xanthan gum powder (obtained from Spectrum Chemical Mfg. Corp. (Gardena, CA)) locust bean gum powder (obtained from Spectrum Chemical Mfg. Corp. (Gardena, CA)) in a 1 : 1 weight ratio with silica to prevent clumping.
• a mold (filamentous fungus), Paecilomyces sp. (3M Culture Designation M- 10) was propagated by aseptically placing one lyophilized pellet of the mold (obtained from
• the inoculated devices were incubated simultaneously at 25°C using an incubation and imaging system as follows.
• the system included an incubation system having platform coupled to a temperature controller (product number TC-36-25-RS232 obtained from TE Technology, Inc. (Traverse City, MI), an illumination source (Leica KL2500LCD 3000 degrees K available from Leica Microsystems GmbH (Wetzlar, Germany), coupled to two illumination guides (Schott A08975 available from Schott North America, Inc. (Southbridge, MA), and a camera (NIKON Coolpix 8400. The camera was positioned about 14 inches above the platform to take images at 60 minute intervals during incubation of the thin film culture devices.
• the elapsed time for detection on the device of Example 1 was determined by observing the light scatter through the device and noting the earliest time a change in scatter was detected, as indicated by clearing zones Figs 4a and 4b.
• the elapsed time for the control device was determined by observing the color change of the indicator in the device at the earliest time the change was detected. Observations from the images for Paecilomyces sp on the devices of Example 1 and the control are shown in Table 2. The observations note the visual changes of the images at the time elapsed since inoculation. The earliest time at which the visual perception threshold is reached for identifying a colony forming unit (CFU) is noted as well as continuing changes throughout the incubation period. The elapsed time for the earliest detection for all of the plates is summarized in Table 3.
• control plates used were 3M PETRIFILM Aerobic Count plates, obtained from 3M Company (St. Paul, MN).
• Two bacterial organisms obtained from the American Type Culture Collection (Manasses, VA) were used to evaluate the culture device of Example 1 and a control.
• the organisms were Escherichia coli (ATCC 51813) and Staphylococcus aureus (ATCC 25923).
• Bacterial cultures were prepared by inoculating pure cultures into tryptic soy broth (TSB available from Remel (Lenexa KS) and incubated at 35°C for 21 hours, static.
• TTB tryptic soy broth
• One loop (about 5 microliters) was transferred into fresh TSB and incubated at 35°C for 21 hours, static.
• 10 microliters of the vortexed culture pipetted into 99 mL of
• BPD Butterfield's Phosphate Diluent
• CFU colony forming units
• PETRIFILM Aerobic Count plate according to the manufacturer's instructions. One milliliter was used to inoculate devices of Example 1 in the same manner. Inoculated plates were incubated at 35°C for 48 hours and imaged using the system and method described in Example 3. Images were taken at 30 minute intervals. The elapsed time for observing the first CFUs is summarized in Table 4.
• a culture device was prepared according to the method of Example 2 and tested according to the method of Example 4 using Escherichia coli (ATCC 51813).
• a control device was also prepared and tested. The culture devices were incubated at 35°C for 48 hours.
• the device of Example 2 with E. coli was placed on a reflector (VikuitiTM Enhanced Specular Reflector. The colonies were noticeable more apparent to the naked eye when the device was observed on the reflector.
• the reflector background made the CFU morphology easier to see because of the enhanced contrast. There was also better contrast between the bubble and bubble-free zones.
• a thin film culture device was prepared according to the method of Example 2 except that the gum used was a modified guar powder (Jaguar CI 62 obtained from Rhodia).
• Thin film culture devices and constituent parts were measured for optical properties - light transmittance, haze, and clarity.
• the % haze is an indication of the amount of light scattering and is an indication of how easy it is to observe changes in the CFU morphology during incubation.
• Measurements for haze, clarity and transmittance were measured in the transmission mode according to the manufacturer's instructions and in conformance to ASTM 1003 using a light measuring instrument (BYK Gardner Haze-gard plus, Catalog #4725, Serial #102485 available from BYK-Gardner USA (Columbia, MD).
• a baseline for the instrument was established without a device under test, i.e., the tape or the thin film culture device, etc. in the sample path and results were 0% haze, 100% clarity, and 100% transmission as expected.
• Sample 1 was a dry powder coated cover sheet from a 3M PETRIFILM Aerobic Count plate (AC plate) tested with the powder side facing the light detector sphere.
• Sample 2 was two cover sheets from AC plates inoculated with a buffer.
• Sample 3 was a biaxially oriented polypropylene (BOPP) film without adhesive from an AC plate.
• Sample 4 was the BOPP film with the adhesive from an AC Plate.
• Sample 5 was 3MTM Advanced Polyolefin Diagnostic Tape Catalog # 9795R.
• Sample 6 was the base member coupled with the cover sheet from Example 2 and inoculated with liquid diluent.
• Sample 7 was the device of Example 6 inoculated with a liquid diluent.
• Example 9 The powders for the cover sheets of Examples 1 and 2 are sterilized by treatment with ethylene oxide gas and then aerated to remove any residual ethylene oxide gas prior to coating.
• Example 9 The powders for the cover sheets of Examples 1 and 2 are sterilized by treatment with ethylene oxide gas and then aerated to remove any residual ethylene oxide gas prior to coating.
• Thin film culture devices of Examples 1 and 2 are placed in an ethylene oxide atmosphere in an enclosed chamber and sterilized.

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