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
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/06—Plates; Walls; Drawers; Multilayer plates
• • 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
  • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
• • 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
  • C12Q1/045—Culture media therefor

Definitions

• This invention is concerned with apparatus for determining the presence of microorganisms which can thrive in anaerobic conditions, particularly sulphate- reducing bacteria.
• Sulphate-reducing bacteria are a group of chemoautotrophic organisms which are able to utilise inorganic sulphate as an energy source, reducing the sulphate in the process.
• Sulphate-reducing bacteria may be found in a variety of places, for example in gas and oil installations, oil products, paper mills and the like; in fact these bacteria may be found in any situation where anaerobic conditions, associated with oil/water, exist. Sulphate reduction by these bacteria can result in foul odours, corrosion, and split emulsions. Anaerobic micro ⁇ organisms are therefore of considerable industrial importance and a requirement exists for a simple rapid method, and apparatus, for detecting the presence of such organisms especially sulphate-reducing bacteria.
• the MicroCheck division of The Boots Company PLC supplies several products known as Boots MicroCheck Dipslides, each of which consists of a bottle within which is inserted a cap/blade unit, the cap sealing the bottle.
• the blade unit carries on one side a culture medium suitable for the culture of aerobic bacteria and on the other side a culture medium suitable for the culture of aerobic fungi, of the type which are commonly found in industrial fluids, cooling waters and on surfaces.
• the cap is removed from the bottle withdrawing the cap/blade unit and the culture media are brought into contact with the fluid to be tested or into contact with the surface to be tested.
• the inoculated dipslide is returned to the tube, capped and incubated at a suitable temperature for a suitable period of time which may range from twenty four hours to seven days depending upon the temperatures involved and other factors.
• a suitable temperature for example, colonies of bacteria or fungi appear on the relevant culture medium and the extent of the contamination is assessed by a suitable method, for example by comparing the number or density of colonies with an interpretation chart.
• apparatus for detecting the presence of organisms which may exist in an anaerobic environment, comprising a container, support means removably assembled with the container to provide a sealed enclosure and comprising within the enclosure a culture medium for the organisms, and generator means within the enclosure which can be activated to generate anaerobic conditions within the enclosure.
• any suitable culture medium for the organisms to be detected may be used and a number of appropriate culture media are known.
• the culture medium is adapted to isolate and identify sulphate-reducing bacteria; preferably the culture medium consists of an iron sulphite agar, a preferred composition of which consists essentially of 10 g/1 tryptone, 0.5 g/1 sodium sulphite, 0.5 g/1 iron citrate, 20 g/1 agar and 20 g/1 of gelatin, and has a pH of 7.1 ⁇ 0.2.
• Any suitable anaerobic generator means may be used and a number of generator means are known.
• a preferred generator means which has been found to be especially effective comprises a mixture of 4 to 8 parts by weight iron metal powder, 1 part by weight citric acid powder and 1 part by weight sodium bicarbonate powder.
• apparatus in accordance with the invention comprises an indicator, for example a methylene blue or resazurine anaerobic indicator, within the enclosure, which can indicate the presence of anaerobic conditions within the enclosure; suitably the indicator is carried by the support means.
• an indicator for example a methylene blue or resazurine anaerobic indicator, within the enclosure, which can indicate the presence of anaerobic conditions within the enclosure; suitably the indicator is carried by the support means.
• the container is a bottle, flask, vial, tube or the like which has an open neck at one end; a preferred container is generally tubular having an open neck at one end thereof, and which is closed at the other end.
• the support means comprises a blade portion on which the culture medium is carried, suitably in two recesses one on either side of the blade portion, and a closure portion from which the blade portion projects.
• the closure portion is adapted to close the neck with the blade portion within the container to provide a sealed enclosure, and suitably comprises a bung adapted to be slidingly received in the neck.
• a cap is adapted to be positioned over the neck and bung; the bung comprises a handle portion which projects through an opening in the cap.
• apparatus in accordance with the invention further comprises a housing for the generator means, the housing suitably comprising a cup which, in use, is positioned in the container.
• the generator means is retained in the cup by a porous cover.
• the housing is positioned in the container at the end portion remote from the neck. The housing may be placed in the container during manufacture or it may be inserted into the container immediately prior to use.
• the container itself comprises a housing for the generator means and suitably the housing is made as a separate part which is assembled in sealing engagement with a tubular body to provide said container.
• the housing has an open neck and a removable cap adapted to be positioned over said neck and the generator means is retained within the housing by a porous cover.
• the cover is water-resistant but gas-permeable.
• Another aspect the invention provides a method of detecting the presence of microorganisms which may exist in an anaerobic environment comprising providing apparatus in accordance with the invention, bringing the culture medium into contact with the surface or fluid environment to be tested to introduce organisms to the culture medium if any are present, activating the generator means and assembling the support means and container to provide a sealed enclosure and anaerobic conditions within the enclosure, subjecting the culture medium to a suitable temperature for a sufficient time to incubate organisms if any are present, and visually inspecting the culture medium after an appropriate time to detect the presence of organisms, if any.
• FIG. 1 is a front view of apparatus embodying the invention
• Figure 2 is a side view of the apparatus shown in Figure 1 ;
• Figure 3 is an end view of the apparatus shown in Figures 1 and 2;
• Figure 4 is a side view of a preferred apparatus embodying the invention.
• Figure 5 is an exploded side view of the apparatus shown in Figure 4.
• the illustrative apparatus comprises a container, namely a cylindrical tube 10, suitably moulded of transparent plastics material, which is closed at one end while having an open neck at the other end.
• the neck is normally closed by support means removably assembled with the tube 10 to provide a sealed enclosure within the tube 10.
• the support means comprises a closure portion including a bung 12 adapted to be slidingly received in the neck of the container 10, a blade portion 14 projecting from the bung 12 into the container, substantially on the diameter of the tube 10 and a handle portion 16 projecting outwardly from the bung 12.
• the apparatus further comprises a cap 18 adapted to be received over the neck of the tube 10 and to snap into sealing engagement over a rim 20 round the neck of the tube 10.
• the cap 18 has a central aperture through which the handle portion 16 projects.
• the cap 18 is effective to retain the bung 12 in the tube 10 and to assist in sealing the enclosure. It will be understood that the material of both the plastics tube 10 and the bung 12 is impervious to air and moisture.
• the tube ' 10 is of clear plastics material, conveniently a polystyrene material. It is suitably about 115 mm long and about 32 mm outside diameter.
• the cap 18 is of a more flexible plastics material e.g. polyethylene.
• the blade portion 14 comprises a panel 22 adjacent the bung 12, the panel 22 being provided with a suitable anaerobic indicator protected by a peelable cover, for example a methylene blue or resazurine anaerobic indicator.
• the panel 22 is connected to the remainder of the blade portion 14 by an integral flexible plastic hinge portion 24 which allows the remainder of the blade portion 14 to be pivoted about the hinge portion 24 so that it can be angled relative to the plane of the panel 22.
• the remainder of the blade portion 14 is provided on either side with recesses 26 which contain a suitable culture medium 28.
• an extension 30 is provided at the end of the blade portion 14 remote from the bung 12 .
• the blade portion 14 is moulded of a suitable plastics material e.g. polypropylene.
• a housing for anaerobic generator means is provided by a plastics cup 32 received in the tube 10 adjacent the closed end thereof.
• the cup 32 contains anaerobic generator means 34 and is covered by a porous cover sheet 36.
• the cup 32 may be placed in the tube 10 during manufacture or may be inserted into the tube 10 immediately prior to use.
• the cup 32 is stored outside the tube 10 prior to use it is preferably provided with a removable, moisture impervious seal (not shown in the drawings) which is removed before the cup is inserted into the tube 10.
• the extension 30 is located adjacent the cover sheet 36.
• the cap 18 enhances both safety (it is impossible to accidentally withdraw the bung 12 with the cap 18 in place) and shelf-life (the cap impedes moisture loss thus prolonging the shelf-life of the apparatus) .
• the culture medium 28 used in the illustrative apparatus is an iron sulphite agar of the following composition:- 10 g/1 tryptone, 0.5 g/1 sodium sulphite, 0.5 g/1 iron citrate, 20 g/1 agar and 20 g/1 gelatin; the pH of the culture medium is 7.1 + 0.2.
• Each of the pockets 26 includes about 4 millilitres of the culture medium 28.
• the anaerobic generator means 34 used in the illustrative apparatus is a mixture containing 4 to 8 grams iron metal powder, 1 gram citric acid powder, and 1 gram sodium bicarbonate powder.
• the illustrative apparatus is supplied in the closed sealed condition in which it is shown in Figures 1 and 2.
• the cap 18 is removed by easing it over the rim 20 at the neck of the tube 10 and the support means is withdrawn from the tube, taking care not to touch the culture medium 28 whilst removing the blade portion 14 from the tube 10 and also ensuring contact with other surfaces is avoided.
• the blade portion is then dipped into fluid to be tested, immersed to the end of the pocket adjacent the hinge portion 24 (care being taken to avoid fluid contacting the panel 22) or brought into contact with fluid in some other way, for example by spraying the fluid onto the culture medium 28; alternatively, if a surface is to be tested, the blade portion is pressed onto the surface in flat face-to-face contact with the surface to be tested.
• the hinge portion 24 allows the blade portion 14 to flex so that the culture medium can easily be placed against a surface to be tested.
• the generator means 34 is activated.
• the generator means 34 is activated by adding 3 to 4 millilitres of distilled water through the porous cover sheet 36 of the cup 32. This will activate the generator means 34 to generate an anaerobic atmosphere within the enclosure defined by the tube 10 and bung 12.
• the cover sheet is then peeled from the panel 22 exposing the indicator. If necessary, the indicator may be moistened to activate it after the cover sheet is removed. Finally the blade portion 14 is replaced in the tube 10 and the bung 12 carefully inserted in the neck, the cap 18 finally being snapped in place over the rim 20 of the neck of the tube to fully seal the enclosure.
• any organisms on the culture medium 28 are incubated at a suitable temperature for a suitable time.
• a temperature of about 32°C for a period of up to 7 days may be appropriate.
• the indicator on the panel 22 is checked visually, through the transparent tube 10; where, for example the indicator used is methylene blue the indicator should remain colourless, confirming anaerobic conditions within the enclosure bounded by the tube 10 and bung 12.
• the culture medium 28 is also inspected to detect the presence of organisms, if any.
• the medium may be inspected visually through the transparent tube 10 for colonies of bacteria on the surfaces of the culture medium 28.
• the presence of black-coloured colonies on the culture medium 28 indicates the presence of sulphate-reducing bacteria.
• Other colonies may develop which are white in colour: these strict or facultative anaerobes are not sulphate-reducing bacteria.
• the level of contamination of the fluid or surface which has been tested can be determined by counting the number of black-coloured colonies on the surface or by comparing the appearance of the surface with a suitable interpretation card which is supplied with the illustrative apparatus and has a number of panels representative of the surface of the culture medium 28, indicating its appearance at various degrees of contamination: thus the amount of contamination can be estimated by comparing the surface of the culture medium 28 with the various depictions on the interpretation card.
• the apparatus should be decontaminated, for example by incineration, autoclaving or disinfection.
• the illustrative apparatus and its method of use are appropriate for various industrial fields, for example the gas and oil industry, the checking of cutting oil fluids in the metal working industry, paper mills, sewage sludge, paint, and industrial water systems.
• the illustrative apparatus is compact, convenient and readily portable and is found to give a reasonably reliable determination of the contamination level of the various fluids and surfaces and has an acceptable shelf-life. Also the use of a transparent tube 10 enables the visual inspection of the indicator and of the culture medium to be performed without the need to remove the bung 12 from the tube 10.
• the interior of the tube 10 and the support means, including the bung 12 and the blade portion 14, as well as the cup 32 and the culture medium 28 and generator means 34 are all sterile when supplied thus reducing the likelihood that false readings will be obtained.
• the container comprises a cylindrical tube 10 e.g. of clear polystyrene which is assembled with a separately made housing 38 e.g. of polyethylene.
• the housing 38 has an open neck at the end remote from the tube 10, the neck being normally closed by a removable cap 40 e.g. of polyethylene. It will be understood that when the housing 38 is assembled with the tube 10 they are sealed together to provide the container and with the bung 12 in place form a sealed enclosure.
• the housing is provided with a water-resistant gas-permeable cover 42 adjacent the tube 10 which is conveniently supported by an annular washer 44.

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• 1988
  • 1988-08-31 GB GB888820594A patent/GB8820594D0/en active Pending
• 1989
  • 1989-08-30 AU AU42092/89A patent/AU4209289A/en not_active Abandoned
  • 1989-08-30 WO PCT/GB1989/001006 patent/WO1990002169A1/en not_active Application Discontinuation
  • 1989-08-30 EP EP19890910124 patent/EP0431056A1/de not_active Withdrawn
• 1991
  • 1991-02-26 GB GB9104009A patent/GB2241504B/en not_active Expired - Lifetime

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