EQUIPMENT FOR DILUTION TECHNIQUE IN MICROBIOLOGICAL ANALYSES

Abstract: Equipment for dilution technique in microbiological analyses comprises a number of mutually separated dilution chambers and a device for the fixation of the chambers, which are closed disposable containers (1) of soft plastics interconnected along a perforated weld (6). Each chamber (1) contains a well-defined quantity of diluent (2), and each chamber may upwardly be provided with an opening (5) through which fluid may be introduced and removed. The equipment is comparable to traditional equipment based on the test tube method in view of reliability, it is economic in view of staff and time because cleaning and autoclaving of glassware are avoided.

Description: Equipment for Dilution Technique in Microbiological Analyses The present invention concerns equipment of the type stated in the introductory portion of claim 1 for use in dilution technique in connection with microbiological analyses. In microbiological analyses of e.g. food products, the food product to be analyzed is usually diluted so that the number of microorganisms is decimated to a number which it is possible to count. If it is a solid material, the dilution process is commenced by making a mechanical homogenization in e.g. a Stomacher ^ apparatus. By means of sterile pincers or a similar implement a known number of grams of the sample material is transferred to a ste- rile bag, e.g. a Stomacher ^ plastics bag. To the sample is added a quantity of diluent, most frequently in the form of an aqueous sodium chloride solution, optionally with the addition of peptone, which equals a dilution of the material of 10 times. The diluted material, the concentration of which is 10 compared to the original concentration, is subjected to the above-mentioned homo- genization in a Stomacher ^ apparatus. By means of a sterile pipette 1 ml of the diluted sample is transferred to a medium-sized test tube, which already contains 9 ml of sterile diluent. Mixing is performed with a new sterile pipette, the mixture being sucked 10 times into the pipette and returned to the test tube. _2 From the resulting dilution, whose concentration is 10 compared to the original concentration, is now trans¬ ferred 1 ml of dilution with the same pipette to a new test tube which already contains 9 ml of diluent. Upon _3 mixture a diluent appears whose concentration is 10 compared to the original concentration. By an analogous working procedure the desired number of diluents is prepared. From the diluents 1 ml or 0.1 ml is taken with a sterile pipette for pour plating and surface plating respectively in various substrates. This well-known dilution technique requires availability of e.g. sterile diluent for the making of the first di¬ lution (10~ ) as well as a number of sterile test tubes, each containing 9 ml of sterile diluent. Due to the ac¬ curacy of the analysis it is extremely important that the 9 ml diluent in each test tube has been measured very carefully, and the production of the mentioned di¬ luents is time consuming. Moreover, the known procedure requires that such apparatus as analysis balance and autoclave are available, and the procedure also requires the presence of a staff who are able to work under sterile conditions and with great accuracy. To the time used for performing the analysis based on dilution technique must be added the time (plus staff and equipment) required for washing and sterilization of glassware and sterili¬ zation of the diluent. The object of the present invention is to remedy the above-mentioned disadvantages by providing equipment by which it is possible to perform microbiological ana¬ lyses on-the basis of traditional dilution technique without the presence of traditional glassware. Instead of the required number of test tubes containing sterile diluent the equipment according to the invention comprises a corresponding number of mutually separated diluent chambers for the production of successive dilu¬ tions and a device for the fixing of the chambers, and it is characterized in that the diluent chambers are closed containers of soft plastics which are intercon¬ nected along a perforated weld, and which each contain a well-defined quantity of diluent, as each chamber may upwardly be provided with an opening through which fluid may be introduced and removed. The chambers, each of which typically contains 9 ml of sterile diluent, are mutually interconnected along a perforated weld according to the same principle as e.g. waste bags of plastics which are available in rolls. In this way the chambers may be separated very easily and quickly, as it is possible beforehand to tear off the number of chambers which it is contemplated to use in the analysis in question, in principle from an endless row of coherent chambers. Various embodiments of containers for various biological purposes are known. Thus the US Patent Specification No. 3 036 894 describes coherent containers of the plas¬ tics bag type, in which each such chamber (sub-chamber) has been designed to contain a certain amount of diluent. The sample to be analyzed is added to the first chamber and moved as a sub-quantity from chamber to chamber through a connection activated by pressure between the chambers so as to produce successive dilutions. However, the object is not primarily to produce dilutions, but, on the con¬ trary, to perform various analytic reactions especially . for the examination of blood tests. The DK Patent Specification No. 140 864 describes an analytic "test package" with reaction chambers and reagent chambers which are arranged in such a way that the reagent chamber supply reagents to the reaction chamber, to which moreover a sample to be tested may be added externally. The sample is added by means of a perforation pipette. The equipment is used especially for photometric analy- ses of urea, glucose, enzymes etc. A heat treatment of such "test packages" is described in the DE Auslegeschrift No. 2 002 218. The GB Patent Specification No. 968 580 mentions single packages in the shape of separate bags containing specific quantities of a diluent. The bags are individually provided with a stopper and intended for the later handling with the test pipettes. The equip¬ ment is preferably used in connection with blood tests. The SE Published Application No. 412 126 describes a disposable device consisting of containers with a pre¬ determined quantity of reagent and with a perforation closure. The containers are handled with a disposable pipette tube for the taking of samples. The device is used e.g. for test taking and dilution of fluids espe¬ cially biological fluids. An apparatus for the filling of chambers is described in the GB Patent Specification No. 1 216 172, showing closed containers placed in a row on a conveyor. These containers are intended for use in equipment for e.g. treatment of bacteria cultures harmful to humans. A set of closed containers containing various quantities of a reaction agent and to which samples for analyzation are injected is described in the US Patent Specification No. 4 391 780. The containers are useful for comparative examinations of a test substance. Known is also disposable equipment comprising coherent chambers for use in culturing microorganisms and produc¬ tion of nutrient media. Such types of disposable equip- ment are e.g. mentioned in the US Patent Specification No. 3 102 08 * 2 and in the DE Auslegeschriften No. 1 283 440 and No. 1 205 231. However, disposable equipment for purposes of dilution technique in microbiological analyses is not known. The invention will be explained in more detail below with reference to the drawing, in which fig. 1 is an outline of a typical embodiment of the equip¬ ment according to the invention, fig. 2 is an outline of another embodiment of the equip¬ ment according to the invention, and fig. 3 shows a possible placing of the dilution chambers in a rack. The equipment according to the invention comprises, as mentioned, a number of mutually separated diluent chambers made of soft plastics, each containing a well-defined quantity of sterile diluent, preferably 9 ml of a suit¬ able fluid, such as an aqueous NaCl-solution with or without the addition of peptone. The shape and the size of the individual chambers are not critical; however, for practical reasons a rectangular shape with a size corresponding to the volume of medium-sized test tubes will frequently be chosen. Fig. 1 shows an outline of a typical embodiment of a number of rectangular diluent chambers (1) interconnected along the long sides at a perforated weld (6), and which each contain a well-defined amount of diluent (2). At the top of each chamber an opening (5) may be made, through which fluid may be supplied and removed by means of e.g. a perforation pipette. The coherent row of chambers may be fixed at its upper (4) and lower (3) closed edge to a rack (not shown in the figure), serving for suspension of the chambers. The rack replacing the conventional test tube rack is provided with clips for the fixation and stretching of the chambers. Moreover, it is provided with a rocking device so that the chambers may be swayed away from a vertical position. Fig. 2 shows another appropriate embodiment of a number of rectangular diluent chambers (1), where the chambers are interconnected along the short sides at a perforated weld (6). The chambers, each containing a well-defined quantity of diluent (2), may be suspended individually in a lug (7) in the form of a melted ring whose centre, which is separated from the inner core of the chamber, may be pierced. Upon suspension, which is shown in fig. 3, an opening (5) at the top of the chamber may be pro¬ duced by cutting one corner off. The material which is used for the production of the coherent diluent chambers may be selected from a number of ordinary plastics materials. For practical reasons, soft, preferably transparant plastics types are used, as the chambers can then be produced as rolls for tearing off. The exact type of plastics will depend on the con¬ templated embodiment and the type of the sterilization treatments to which the chambers should be submitted during production. The plastics material may e.g. be sterilized by means of electronic irradiation or by means of ordinary autoclaving. No matter which of the two above-mentioned sterilization methods is used or perhaps a third one, the demands on the plastics material should be that its qualities in view of water vapour permeability and its strength in view of ultimate strength and welding strength are not deteriorated at the treatment. As examples of soft plas¬ tics materials whose qualities are not deteriorated by electronic irradiation may be mentioned polyethylene, polyethylene with ethylene vinylacetate and polyamide polyethylene laminates, see K. Nielsen "Elektronbestra- ling af emballager", Levnedsmiddelbladet No. 5, May 1985. Such materials have been subjected to electronic irradi¬ ations of 10 and 30 kGy, and the measurements did not show any decisive changes with respect to the above-men- tioned qualities. For autoclaving, e.g. polyester sheet may be applied. This material is also used for the pro¬ duction of cooking bags, see Leif Bøgh-Sørensen et al. "Konserveringsteknik", DSR-forlag, Copenhagen, 1978. The invention is illustrated more fully by the following example: EXAMPLE With the purpose of examining whether the method according to the invention with irradiation-sterilized diluent rows "embedded" in polyethylene differs from the tradi- tional method, where the diluent material is applied to test tubes which are then autoclaved, both methods have been used for the determination of total count of chicken neck skin. Production of Diluent Chambers of Polyethylene Diluent chambers are made of polyethylene, and to the chambers were added 9 ml of an 0.9% NaCl-solution and 9 ml of an 0.9% NaCl-solution containing 0.1% peptone. Upon injection of the solution the chambers were closed hermetically by means of a weld. The diluents embedded in plastics were sterilized by irradiation at Raychem A/S, Glostrup, the irradiation dose used being approx. 30 kGy. Production of Traditional Diluents Medium-sized test tubes are injected with 9 ml of 0.9% NaCl-solution and 9 ml of 0.9% NaCl-solution containing 0.1% peptone. The test tubes are closed by means of a cotton wool stopper, upon which they are placed in a wire rack and autoclaved. Plating from Diluent Rows 10 g of chicken neck skin, kept at 5°C for 8 days, are transferred to a Stomacher w bag, upon which 90 ml of 0.9% NaCl-solution with 0.1% peptone are added. The di- luent is homogenized in a Stomacher ^ apparatus. The subsequent dilutions are made in irradiated diluent rows of plastics containing a pure NaCl-solution and an NaCl-solution with peptone, respectively. The top of each plastics chamber is cut off, and into the first chamber is injected 1 ml of the homogenized mixture, upon which the solutions are mixed in the chamber by carefully pressing it with the fingers. To each of the following chambers is injected 1 ml of the mixture from the preceding chamber, and in each case mixing of the fluids is performed by pressing the plastics bags care¬ fully. Analogously, dilutions are made according to the tradi¬ tional principle with autoclaved diluent rows in test tubes. From 10 and 10 dilutions, plating on agar are made by using the pour plate method, upon which they are incubated at 20°C and read after 4 nights and days. Moreover, sterilization control on the various solutions is effected. Results Number of colonies counted on agar after 4 nights and days at 20°C. Diluent Sterilization Diluent solution 10 10 -7 control Irradiated NaCl solu¬ approx. tion in polyethylene 240 23 Autoclaved NaCl solu¬ approx. tion in test tubes 270 23 Irradiated NaCl + P approx. solution in polyethylene 300 28 Autoclaved NaCl + P approx. solution in test tubes 240 18 As appears from the above results, all tests are in the 8 8 range from 2 x 10 to 3 x 10 total count per g chicken neck skin, and it may thus be concluded that the relia¬ bility of using irradiated diluent rows in polyethylene is fully comparable to the technology based on the tradi¬ tional test tube methods.

Claims: P a t e n t C l a l m s : 1. Equipment for dilution technique in microbiological analyses, comprising a number of mutually separated di¬ luent chambers for the production of successive diluents and a device for the fixation of the chambers, c h a ¬ r a c t e r i z e d in that the diluent chambers are closed containers (1) of soft plastics, which are inter¬ connected along a perforated weld (6) and which each contain a well-defined quantity of diluent (2), as each chamber may upwardly be provided with an opening (5) through which fluid may be introduced and removed. 2. Equipment according to claim 4, c h a r a c t e r ¬ i z e d in that the plastics material is selected among polyethylene, polyethylene together with ethylene vin- cylacetate and polyamide polyethylene laminates. 3. Equipment according to claim 1 or 2, c h a r a c ¬ t e r i z e d in that the plastics material used is selected among materials whose qualities in view of water vapour permeability and strength by way of ultimate strength and welding strength are not deteriorated by the sterilization process. 4. Equipment according to any of the preceding claims, c h a r a c t e r i z e d in that the diluent is an aqueous sodium chloride solution to which the peptone is optionally added. 5. Equipment according to claim 1 or 4, c h a r a c ¬ t e r i z e d in that the volume of the diluent in each chamber is 9 ml . 6. Equipment according to any of the preceding claims. c h a r a c t e r i z e d in that the opening (5) , through which fluid may be introduced and removed, is an opening made by a penetration pipette, or an opening which has been cut after the placing of the diluent cham¬ ber in a rack.

Inventors: SVENDSEN FINN ULRIK, SVENDSEN, FINN, ULRIK
Applicants: SVENDSEN FINN ULRIK, SVENDSEN, FINN, ULRIK
Application year: 1987
Main classification: C12M1
IPC codes: C12M1