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
  • C12Q1/06—Quantitative determination
• • 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
  • C12M23/10—Petri dish
• • 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
  • C12M23/20—Material Coatings
• • 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
  • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
  • C12M33/02—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by impregnation, e.g. using swabs or loops
• • 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

Definitions

• the present invention relates to organism culturing methods and articles, and more particularly to a device for spreading a liquid suspension of microorganisms on the surface of a culture medium.
• the invention is particularly applicable to enumeration of microorganisms in water, foods and the like, and will be described with particular reference thereto although it will be appreciated that the invention has other applications.
• Enumeration of microorganisms in water, foods, pharmaceuticals, etc. requires the application of samples to a culture medium, such as a petri plate containing mixtures of nutrients and agar, wherein individual organisms from a sample multiply on the surface of the agar to form visible colonies which are then counted.
• a culture medium such as a petri plate containing mixtures of nutrients and agar
• individual organisms from a sample multiply on the surface of the agar to form visible colonies which are then counted.
• To ascertain the number of organisms in a test sample it is conventionally known to prepare a series of dilutions of the original sample and then apply 0.1 ml of each dilution of the surface of a
• ** culture medium i.e. the agar.
• a spreading device typically a sterilized bent glass rod, is used to spread the 0.1 ml of bacterial suspension over the surface of the agar. It is important that the sample be spread as evenly as possible over the agar, in that the more evenly the bacterial suspension is spread on the agar surface, the more information can be derived from each plate. A puddle of cells on one part of the plate results in clusters of colonies which are hard to count and likely to overlap with each other. In this respect, the number of colonies per standard (10 cm) petri plate should be above 30 (for statistical significance) and below 300 (above which overlapping colonies become more likely) . Consequently, the most accurate and reliable information will be obtained when the liquid samples are uniformly spread across the surface of the agar.
• the present invention solves these and other problems, and provides a device and method for spreading a liquid suspension on the surface of a culture medium, which method and device are simple and easy to use and provide an even distribution of the suspension along the surface of the medium.
• a device for uniformly spreading a liquid suspension of bacteria along the surface of a sterile culture medium is comprised of a sterile, flat, non-absorbent fabric having a shape commensurate with the surface profile of the culture medium to lie in registry therewith.
• the fabric is comprised of relatively fine strands disposed relative to the surface of the medium such that a cooperative capillary attraction between the surface of the culture medium and the strands on the liquid suspension is produced and effects an even distribution of the suspension along the surface of the culture medium.
• the invention is generally for use in circular petri plates containing mixtures of nutrients and agars.
• the invention is preferably comprised of a circular piece of nylon netting which is sterile and is placed on the surface of the agar plate on which the bacteria are to be spread.
• An appropriate volume of liquid containing the bacteria is applied to the netting, which serves to spread the liquid over the surface of the plate by capillary action. After the liquid soaks into the plate, the netting is removed, and the bacteria are allowed to grow into colonies in the usual manner.
• the netting uniformly spreads the liquid suspension thereby forming an even layer of liquid. This provides a more evenly spaced or spread colony formation which allows more reliable counting of
• the netting eliminates the need for manually spreading of the suspension on the surface of the culture medium thereby reducing labor costs.
• the fabric is coated with a substance soluble in the liquid suspension, which substance is to be applied to the surface of the culture medium simultaneously with the spreading of the liquid suspen ⁇ sion.
• An object of the present invention is to enumerate bacteria from a liquid in an accurate and efficient manner
• Another object of the present invention is to provide a device which will spread a liquid suspension aver the surface of a culture medium forming an even layer thereon.
• a still further object of the present invention is to provide a device as described above, wherein manual spreading of the liquid suspension is not required.
• FIGURE 1 is a perspective view of a device in accordance with the present invention in exploded relationship to a culture medium;
• FIGURE 2 is an enlarged partial view of the device depicted in FIGURE 1 showing details thereof;
• FIGURE 3 is a greatly enlarged sectional view of the device shown in FIGURE 1 in operative relationship to the surface of the culture medium.
• the present invention is particularly applicable for enumerating microorganisms in a sample by spreading a liquid suspension of such sample on the surface of a culture medium and will be described with respect thereto, although it will be appreciated that the present invention has other broader uses and applica ⁇ tions.
• enumeration of micro ⁇ organisms in water, food, etc. requires application of samples to a culture medium. Important to the results of such procedure is that the suspension be spread as evenly as possible on the agar surface to ensure even separation of colonies thereon.
• the present invention provides a device and method of spreading liquids over the surface of the medium in an even layer without the need for the traditional spreading method.
• FIGURE 1 shows a flat, screen-like element 10 which is provided to lie on a surface 12 of a culture medium 14.
• Element 10 is an open fabric or screen or netting comprised of relatively fine strands 16 with openings 18 there ⁇ between, as seen in FIGURE 2.
• element 10 When placed on culture medium 14, element 10 provides surface area opposed to surface 12 of medium 14.
• liquid When liquid is placed on the medium surface 12, liquid contacts the surface area of strands 16 of element 10 to produce a capillary effect which spreads the liquid along strands 16 of element 10 and along surface 12 of medium 14.
• the screen-like element is preferably shaped commensurate with the profile of the culture medium such that the liquid is spread to the edge of the screen-like element. It will of course be appreciated that the shape of the screen ⁇ like element may assume any configuration which is positionable on the culture medium.
• screen-like element 10 is comprised of tulle which is a commercially available nylon netting with about 11 squares per centimeter which is a mesh width of approximately. 1 millimeter. Circles of the tulle, cut or otherwise formed to match the surface profile of the agar in the petri plate, are sterilized by autoclaving. The tulle circles are then applied in a sterile fashion to the surface of agar plates.
• the agar plates are of sufficiently low moisture content such that the volume of liquid to be applied (for example, 1 ml) will soak into the agar within a convenient period of time (for example, 30 minutes) .
• a convenient period of time for example, 30 minutes.
• Prepared agar plates sold by GIBCO Laboratories, Life Technologies, Inc. have been found to be particularly suitable for use with the present invention.
• the liquid suspension of bacteria is applied to the agar surface through the tulle, and the suspension spreads over the agar of the tulle, forming an even layer of liquid. When the liquid has soaked into the agar, the dry tulle circle is removed.
• tulle Materials other than the nylon netting (tulle) may be used to perform the spreading functions.
• fiberglass screening has been found to be suitable.
• these materials must be very flat when applied to the agar surface, or they must become flat when the liquid is applied through the netting; otherwise uneven spreading of the liquid will result.
• the netting must allow spreading of the liquid.
• complete contact of the netting with the agar surface does not spread the liquid satisfactorily, in that complete surface contact does not provide passages between the tulle, and the agar through which liquid may pass.
• the tulle netting is not pressed onto the agar surface, but rather rests thereon.
• the light weight of the tulle, and its construction provide openings or gaps 22, best seen in FIGURE 3, which gaps permit passage of the liquid suspension therethrough.
• the tulle or netting is coated with a substance (e.g., an antibiotic) which is soluble in the liquid suspen ⁇ sion.
• a substance e.g., an antibiotic
• the substance can be applied to the culture medium surface simultaneously with the spreading of the liquid.
• the utility of the tulle screen method for enumerating bacteria was demonstrated as follows.
• a sample of Escherichia coli bacteria estimated to contain about 10 8 organisms per ml was prepared.
• a series of dilutions was prepared to give an estimated 30 - 10,000 bacteria per ml. Aliquots of these dilutions were applied in triplicate to agar plates by the traditional spreading method (0.1 ml aliquots) or using screens (1.0 ml aliquots) .
• the bacteria were allowed to grow into colonies and the colonies were counted.
• the concentra ⁇ tion of bacteria in the original preparation was calculated for each aliquot and the results were as follows:
• the netting may be comprised of a material which, after a period of time sufficient to evenly spread the liquid, will dissolve, thereby eliminating the necessity of removing the netting.
• incorporation of a tab or other protrusion from the edge of the screen will facilitate handling of the screens. It is intended that all such modifications and alterations be included insofar as they come within the scope of the claims or the equivalence thereof.

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• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Biomedical Technology (AREA)
• Sustainable Development (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Analytical Chemistry (AREA)
• Clinical Laboratory Science (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Pathology (AREA)
• Toxicology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

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• 1987
  • 1987-05-14 EP EP19870903775 patent/EP0268651A4/de not_active Withdrawn
  • 1987-05-14 AU AU74887/87A patent/AU7488787A/en not_active Abandoned
  • 1987-05-14 WO PCT/US1987/001128 patent/WO1987006955A1/en not_active Application Discontinuation

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