DE2432049A1 - COMPOSITE OF MEMBRANE FILTERS AND POROESE MATERIAL AND ITS USE - Google Patents

Description

Dr Ho / A

C. H. Boehringer Sohn, Ingelheim am Rhein

and
Sartorius membrane filter GmbH

Göttingen, Weender Landstrasse 94-102

Composite of membrane filters and porous material and its use

The invention relates to a composite of membrane filters and porous material, the method for its production and the use as a carrier of nutrient media for microbiological investigations.

Membrane filters are graded according to different permeability Methods and made from different fabrics. They consist e.g. B. from a thin skin of colloidal solutions, z. B. gelatine, collodion, silicic acid, cellulose esters or cellulose, which build up a framework when evaporating and coagulating, the cavities of which form a sponge-like structure. The cross-sections of the pores can be determined by the manufacturing method vary widely, so that e.g. bacteria and even particles up to molecular size can be sieved off.

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Membrane filters normally have to be supported during use, with larger dimensions usually on sieve plates or frits, as they are not mechanical in themselves are sufficiently firm.

For many purposes, a solid composite of membrane filter and a porous base would be very desirable, wherein the properties of the membrane filter on the one hand and the properties of the porous substrate on the other hand should be retained completely and unchanged.

It is known to use adhesive to glue the membrane filter to a porous substrate, but this has the disadvantage that the adhesive can influence the properties of the membrane filter / porous substrate system in an unpredictable manner. It is also known to soak a porous substrate with a solution from which a membrane filter is formed during drying, so that the membrane layer is at least partially formed within the porous material. But the so obtained product has different properties than the individual components. This applies above all to the surface, which no longer has the smooth membrane filter surface required for many jobs, but rather reproduces the structure of the porous base.

If the support is a porous support impregnated with nutrient media and dried, for example a cardboard disc, which is to be used for differentiated bacteriological inhibition tests, a membrane filter is required adjacent to the porous support. It is neither possible to create a bond by gluing nor to use the porous material with the that

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To soak membrane filter forming material, as on the one hand the Adhesive layer influences the diffusion of nutrients between the porous substrate and the membrane filter in an unpredictable way, on the other hand, in the absence of a pure, smooth membrane surface, for example no concentric Hemmhof rings arise during the antibiotic test.

The invention has therefore set itself the task of a To achieve composite membrane filter / porous base in which the properties of the two components are fully preserved remain and the disadvantages of the prior art are avoided.

Such a composite is obtained in that a Membrane filters based on polymeric substances during the gelation of the polymeric membrane formers is brought into contact with the porous substrate and solidified thereon.

This creates the composite of membrane filters described at the beginning and porous base. Such a composite can be used, for example, in bacteriological inhibition tests make not only qualitative but even quantitative statements.

According to a special embodiment, such Composite can be produced as follows:

A solution capable of forming a membrane is applied in a known manner to an endless, highly polished belt. Cellulose nitrate, cellulose acetate, gelatin, PVC, PVA, polyamide or alginate are particularly suitable as membrane formers.

The applied solution is guided through certain climatic zones with the help of the moving belt, e.g. as in

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DT-PS 1 017 596 described. During this process, the solvents evaporate, and as a result, Solid substance a porous flat structure, namely the membrane filter. Between the phase of the solution and the solid The filter goes through a stage in which the polymeric membrane former is present as a gel.

If you bring a porous pad during this stage, E.g. a tape made of cardboard, carefully in contact with this gel, so the filter that is formed adheres firmly to it porous material and solidifies on it. After completion of the manufacturing process, the membrane filter is in place as well as the porous base, although in the composite, clearly recognizable in its previous unchanged form. This is particularly evident in the perfectly flawless membrane surface.

In a corresponding manner, a second membrane can be applied to the other side of the porous material.

The two components adhere very well to one another and the porous base and membrane filter are not separated without external influence. The adhesion can be developed more or less firmly as desired, depending on whether the contact between the porous material and the membrane gel is made more at the beginning or towards the end of the gel state of the membrane-forming material. ^T

The composite according to the invention of membrane filter and porous material, especially cardboard about 1 to 2 mm thick, is particularly suitable for microbiological tests in which the formation of sharply delimited, circular zones of inhibition is necessary or desired. The network is to do this with a usual

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Impregnated nutrient solution for microorganisms and essentially takes over the function of such agents as agar or agar Gelatins, which are commonly used to solidify nutrient media.

The impregnated composite (nutrient card) can be used for microbiological purposes immediately after sterilization. It however, it is also possible to dry the impregnated composite, to cut it into suitable shaped bodies, e.g. circular disks and to be stored in a sterilized and sterile packaging. At the desired time, by moistening with sterile water, quickly a culture medium that can be used in the usual way can be produced. The overgrown nutrient cards can, if necessary, after staining dried and after covering with mil: a film as permanent Document to be retained.

The advantages of the composite according to the invention are particularly evident in those microbiological tests in which allows standardization of the tests through reproducible properties of the nutrient medium.

This is particularly successful when synthetic nutrient media are used, i.e. nutrient media made up of defined compounds are composed and not of otherwise common substance mixtures (peptones, meat extract, yeast extract) and other additives.

Such a bynthetic nutrient medium can be composed as follows be:

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Solution I:

Components

g / liter

L-arginine . 5 H ₂ O 0.523 L-aspartic acid 1.065 L-cysteine 0.485 L-glutamic acid 2.207 Glycocolla " 0.15 L-histidine 0.31 L-leucine 0.787 L-isoleucine 0.525 L-lysine 0.585 L-methlonin 0.298 L-pheny! Alanine 0.33 L-proline 0.345 L-threonine 0.357 L-tryptophan 0.102 L-tyrosine 0.362 L-valine 1.17 NaCl 2.92 KCl 0.298 Na acetate, H ₂ O-free 0.984 Na pyruvate 1.1 Components g / liter Na glycerophosphate 5.2 Mg SO ₄ . 7 H ₂ O 0.049 Adenine 0.009 Guanine 0.009 Uracil 0.011 Aqua dest. 950.0 ml

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Solution II; Components

D-glucose inositol choline-Cl Nicotinamide Thiamine HCl Riboflavin ■ Pyridoxine HCl pantothenic acid calcium salt folic acid Biotin Na glycerophosphate * 5 H p-Nitrophenylpropane triol Pimaricin Aqua dest.

g / liter *)

1.98 0.011 0.014 0.011 0.01 0.001 0.006 0.01 0.009 0.0002 1, OA 0.005 0.1049 50.0 ml

Solution III:

10 Jfige aqueous sodium hydroxide solution

The data relate to the amount of distilled water that is used in total for the preparation of solutions I and II, in the present example 950 ml plus 50 ml.

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preparation

1. The ingredients under I are distilled in 950 ml. Dissolved water and sterilized in an autoclave at 121 ° C for 20 minutes.

2. The glucose, vitamins and growth substances under II are

in 50 ml dist. Dissolved water and sterilized by sterile filtration through a membrane filter, pore size 0.22 / µm.

3. The 10% sodium hydroxide solution is sterilized in an autoclave ^{at 121 ° C. for 20 minutes.}

4. Solution I and II are each heated to 55 ° C, then put together; the pH value is adjusted to 7.2 at 47 ° C. with the aid of solution III.

The amounts of the constituents in solutions I and II can also be chosen differently. For example, you can can be reduced to about half or doubled. In general, however, it is not advisable to To omit an ingredient, for example an amino acid, or to change the pH of the mixture significantly. The usage the media in a standardized test is only possible insofar as there are no deviations essential influence on the growth of the microorganisms and thus on the size of the zones of inhibition arising during the test Has.

The test can be carried out in the following ways:

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The composite impregnated with the nutrient solution (circular disk of appropriate diameter) is placed with the membrane layer facing up in a Petri dish and inoculated with a certain amount of the sample to be examined (if necessary after dilution). Then filter paper sheets of about 4 mm diameter, which are loaded with certain amounts of antimicrobial agents (antibiotics, sulfonamides), are distributed on the membrane and the Petri dish is ^{incubated for about 16 to 24 hours at a certain temperature (e.g. 36 * I 0} C).

If there is a certain drug load on the test paper (with a given, to be determined in the usual way) Germ density a relationship between the diameter of the inhibition zone and the minimum inhibitory concentration of the respective active ingredient. On the other hand, of the active ingredients in use, the maximum achievable or therapeutically permissible blood level is known. It is therefore possible, due to the diameter of the inhibition zone, the therapeutic utility to estimate an antimicrobial agent.

If the test is carried out with standardized nutrient solution under standardized conditions, with the composite according to the invention obtained reproducible results. With known comparable means, for example membrane-coated cardboard according to the German Offenlegungsschrift 2 320 946, this is not possible. Another test method in which nutrient cards according to the invention can be used with particular advantage, is described in the German patent application P 23 Ol 211 and the Dutch application 74 00336.

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Claims (10)

- ίο - 24320A9 Claims ΐΛSolid adhesive-free composite of membrane filters and porous, ..-- ^/ material in the form of two clearly distinguishable layers unchanged in their respective properties, characterized in that a membrane filter based on polymeric substances while the polymeric membrane former is in the gel state with the porous material is brought into contact and solidified thereon. 2. Composite according to claim 1, characterized in that the polymeric membrane former cellulose nitrate, cellulose acetate, Gelatin, PVC, PVA, polyamide or alginate. 3. The composite according to claim 1 or 2, characterized in that the porous substrate is cardboard. 4. The composite according to claim 3, characterized in that the porous support is cardboard made of cellulose fibers. 5. Composite according to one of the preceding claims, characterized in that the porous material with nutrient solution is impregnated. 6. Composite according to one of the preceding claims, characterized in that the porous material with nutrient solution impregnated and dried, 7. The composite according to claim 5 or 6, characterized in that the nutrient solution about 0.25 to 1 g of L-arginine, 0.5 - 2 g of L-aspangic acid, 0.25 - 1 g of L-cysteine, 1 - 4 g of L-glutamic acid, 0.1-0.4 g glycocolla, 0.15-0.6 g L-histidine, 0.4-1.6 g L-leucine, 0.25 -Ig L-isoleucine, 0.3-1.2 G L-lysine, 0.15-0.6 g L-methionine, 0.15-0.6 g L-phenylalanine, 0.2-0.8 g L-propine, 0.2-0.8 g L-threonine, 509884/0582 0.05-0.2 g tryptophan, 0.2-0.8 g L-tyrosin _, 0.5-2 g L-valine, 1.5-6 g sodium chloride, 0.15-0.6 g Potassium chloride, 0.5-2 g sodium acetate anhydrous, 0.5-2 g sodium pyruvate, 3-12 g sodium glycerophosphate pentahydrate, 0.025-0.1 g magnesium sulfate heptahydrate, 0.005-0.02 g adenine, 0.005-0.02 g guanine, 0.005-0.02 g uracil, 1-4 g D-glucose, 0.005-0.02 g inositol, 0.005-0.02 g choline chloride, 0.005-0.02 g nicotinic acid amide, 0.005-0.02 g thiamine hydrochloride, 0.0005-0.002 g riboflavin, 0.003-0.012 g pyridoxine hydrochloride, 0.005-0.02 g calcium pantothenate, 0.005-0.02 g folic acid, 0.0001-0.0004 g biotin, 0.0025-0.01 g of p-nitrophenylpropane triol, 0.05-0.2t pimaricin per liter of water as well as an amount of sodium hydroxide in dilute aqueous solution necessary for setting ptl 7.2. 8. A method for producing the composite according to claim 1 to 7 from a solution which is capable of membrane formation is, applying the solution to a pad, evaporating the solvent and solidifying the so formed Membrane filter, characterized in that after Phase of the solution and before the phase of the solid filter, if the polymeric membrane former is present as a gel carefully brings porous material into contact with the gel and the adhering gel of the polymeric membrane former solidified on the porous material. 9. The method according to claim 8, characterized in that the contact of the membrane filter formed with the porous material between entry of syneresis and before final solidification of the membrane filter, which is through ice flower-like appearances of the undressed Films to recognize takes place. 10. Use of the composite according to claim 1 to I ₁ for the determination of therapeutically applicable antimicrobial agents, characterized in that the with 50988A / 058 2 Nutrient fluid-soaked composite after inoculation with a certain amount of a test sample and after placing circular paper sheets, each loaded with a certain amount of an antimicrobial active ingredient, ^{incubated for about 16 to 24 hours at about 35 to 37 0} C and that one then based on the diameter of the resulting zones of inhibition, taking into account the germ density and the relationship between the diameter of the zone of inhibition and the minimum inhibitory concentration, those therapeutics are determined which have a bactericidal effect against the germs to be combated. 50988A / 0582