CA2150720A1 - Solid culture medium and the method of its production - Google Patents
Solid culture medium and the method of its productionInfo
- Publication number
- CA2150720A1 CA2150720A1 CA002150720A CA2150720A CA2150720A1 CA 2150720 A1 CA2150720 A1 CA 2150720A1 CA 002150720 A CA002150720 A CA 002150720A CA 2150720 A CA2150720 A CA 2150720A CA 2150720 A1 CA2150720 A1 CA 2150720A1
- Authority
- CA
- Canada
- Prior art keywords
- acrylamide
- acrylonitrile
- bis
- methylene
- copolymer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000007787 solid Substances 0.000 title claims abstract description 63
- 239000001963 growth medium Substances 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 44
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 7
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims abstract description 75
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 claims abstract description 43
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims abstract description 41
- 229920001577 copolymer Polymers 0.000 claims abstract description 35
- 235000015097 nutrients Nutrition 0.000 claims abstract description 32
- 244000005700 microbiome Species 0.000 claims abstract description 21
- 239000000758 substrate Substances 0.000 claims abstract description 20
- 230000000977 initiatory effect Effects 0.000 claims abstract description 12
- 239000002609 medium Substances 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 239000002904 solvent Substances 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 238000000746 purification Methods 0.000 claims abstract description 4
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 3
- 239000004159 Potassium persulphate Substances 0.000 claims description 16
- RMGVZKRVHHSUIM-UHFFFAOYSA-L dithionate(2-) Chemical compound [O-]S(=O)(=O)S([O-])(=O)=O RMGVZKRVHHSUIM-UHFFFAOYSA-L 0.000 claims description 16
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 16
- 235000019394 potassium persulphate Nutrition 0.000 claims description 16
- 239000011734 sodium Substances 0.000 claims description 15
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 14
- 229910052708 sodium Inorganic materials 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 11
- 238000005470 impregnation Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 12
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 18
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 15
- 238000011534 incubation Methods 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 8
- 239000000499 gel Substances 0.000 description 8
- 239000011159 matrix material Substances 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 239000000178 monomer Substances 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000009792 diffusion process Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 238000005755 formation reaction Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 230000008018 melting Effects 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000007334 copolymerization reaction Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 230000002906 microbiologic effect Effects 0.000 description 2
- 230000009965 odorless effect Effects 0.000 description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000010526 radical polymerization reaction Methods 0.000 description 2
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 125000000030 D-alanine group Chemical group [H]N([H])[C@](C([H])([H])[H])(C(=O)[*])[H] 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 238000012009 microbiological test Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
This solid nutrient medium for cultivating microorganisms contains a copolymer of acrylonitrile and acrylamide and a liquid nutrient substrate in the following percent-by-weight proportions: copolymer of acrylonitrile and acrylamide: 5.0 - 30.0; nutrient substrate: 70.0 - 95Ø The method of obtaining a solid culture medium includes the purification of acrylamide and N,N'-methylene-bis-acrylamide by recrystalization; preparation of the reactive mix composed of acrylonitrile, acrylamide, N,N'-methylene-bis-acrylamide, an acceptable solvent and an initiating system; polymerization over a period sufficient for the production of a copolymer retaining the form; its rinsing and saturation with water until equilbrium state; permeation with the nutrient substrate, where the components have these percent-by-weight proportions: acrylonitrile 5.0 - 50.0; acrylamide 5.0 - 50.0; N,N'-methylene-bis-acrylamide 0.01 - 0.60;initiating system 0.15 - 0.3;
acceptable solvent residuum.
acceptable solvent residuum.
Description
~21 5~72~
SOLID CULTURE MEDIUM
AND THE METHOD OF ITS PRODUCTION
The present invention relates to a solid nutrient medium for cultivating microorganisms and the method of its production. Solid culture media can be usedin medical practice and in biology.
There exists a method of obtaining a solid culture medium for medical and biological purposes by the copolymerization of an acrylamide and a N,N'-methylene-bis-acrylamide in a sodium chloride solution, at a ratio of accordingly 0.5-40.0: 2.5-12.0, until the formation of a polyacrilamide gel. The latter, containing a maximum amount of water, is rinsed in a sodium chloride solution and saturated with nutrient substrates (SU,- 997,466).
The culture medium thus obtained is used to study the biological properties of microorganisms. However, this culture medium, solid as it is, has a markedly vulnerable surface layer, especially in the course of sterilization, inoculation, and when removing colonies with a loop. Repeated inoculations are possible after rinsing the surface in a sodium chloride solution, which is ill-advised in most microbiological manipulations.
Another method of obtaining a solid culture medium consists in a radical copolymerization of an acrylamide and N,N'-methylene-bis-acrylamide in a sodium chloride solution at a ratio of accordingly 15.0-20.0:0.019-0.132, until the formation of a polyacrilamide gel. The gel is rinsed in a physiological solution and let saturate for 16-20 hours at 50-60C. After that the polyacrilamide gel is saturated with nutrient substrates. The solid culture medium thus produced is used for cultivating microorganisms. It is colorless, elastic and displays good adhesive properties. When inoculated, microorganisms spread over the surface, without growing into it, and are completely removed with the loop. However, during storage in Petri dishes and incubation the liquid tends to exude, the effects ofwhich are not totally liquidated even after lengthy periods of thermostatic treatment. Besides, certain types of microorganisms, particularly mobile forms, fail to show the typical growth of colonies in this culture medium despite the presence of characteristic morphological, biochemical and antigenic properties. This limitates the usage of the above culture medium, as for example, in the diagnostics of contageous diseases.
~, ~ 2 1 50720 Finally, there is the method of producing a solid culture medium using a radical polymerization of acrylamide and N,N'-methylene-bis-acrylamide at a massratio of accordingly 15.0-20.0:0.019-0.132, in a sodium chloride solution, until the formation of a polyacrilamide gel, with subsequent rinsing and saturation and permeation with nutrient substrates. Here radical polymerization takes place in the presence of polyvinyl alcohol at a weight ratio of acrylamide and polyvinyl alcohol of 15.0-20.0: 1.0-3.0 (Ca 1,283,873).
However, the structure of the polyacrilamide gel obtained using this method hinders the diffusion of the ions Na+ and K+ (that are part of most of the knownculture media) from within the polyacrilamide gel to its surface where the microorganisms are located. Consequently, the microorganisms showe poorer culture properties, especially when prolonged cultivation is required (as in the case of microbacterias, fungi, etc.).
Besides, the known solid culture medium is not sufficiently effective by its biological indices.
Here and further on we understand by "biological indices":
(a) Sensitivity which is determined by the maximum dilution of a culture, when every inoculated dish (test tube) shows a certain growth (the result reflects an appropriate dilution, with 10-'-10-9 for all the groups of culture media);
(b) Effectiveness, which is determined by the yield of microbic bodies per 1 ml of culture medium (in billion/ml).
Further, it is necessary to improve the physical-mechanical properties of solid culture media obtained using the known techniques.
The present invention is aimed at bettering the diffusive and physical-mechanical properties of a solid nutrient medium for growing mcrioorganisms, as well as at improving its biological indices.
The set goal is attained by the presence in the solid culture medium of a copolymer of acrylonitrile and acrylamide and a liquid nutrient substrate with the following ratios (% by weight):
copolymer of acrylonitrile and acrylamide - 5.0-30.0 liquid nutrient substrate- 70.0-95Ø
(,A21 5~720 The aforementioned nutrient medium for cultivating microorganisms possesses high diffusive and physical-mechanical properties, as well as significant biological indices, which ensures it a number of advantages as compared to the known solid culture media.
The set objective is also attained by the previous purification of the acrylamide and N,N'-methylene-bis-acrylamide by recrystalization as part of the present method of obtaining a solid culture medium; also by the preparation of areactive mix containing acrylonitrile, acrylamide, N,N'-methylene-bis-acrylamide, and acceptable solvent, an initiating system; by the process of polymerization over a period necessary for obtaining the copolymer retaining the form; by the rinsing of the copolymer and its saturation in water and permeation with a liquid nutrient substrate, with the following ratios of components in the reactive mix (% by weight):
acrylonitrile - 5.0 - 50.0 acrylamide - 5.0 - 50.0 methylene-bis-acrylamide - 0.01 - 0.60 initiating system - 0.015 - 0.3 acceptable solvent - the remainder The above method of obtaining a solid culture medium ensures the latter's higher diffusive properties and as a result a better diffusion of the ions Na+ and K+
(as components of most of the known solid culture media) from within the solid nutrient medium to its surface where the microorganisms are located. This, in turn, makes it possible to promote the culture properties of the microorganisms of prolonged cultivation.
Besides, the present method allows to improve the biological and physical-mechanical indices. This advantage is secured by the preparatory purification ofthe acrylamide and N,N'-methylene-bis-acrylamide by recrystalization, whereby all traces of acrylic acid are removed (and further on we show that this acid is present in all the monomers under study, to the amount of some 0.2% by weight of the agent - irrespective of the manufacturing company - and that its presence negatively affects the end product).
Finally, the positive effect of the said method is also achieved by the usage of the purified monomers acrylamide and ~, A21 5~720 N,N'-methylene-bis-acrylamide in the reactive mix of the present composition.
Potassium persulphate and sodium metabisulphate are recommended as the initiating system, in the following proportions (% by weight):
potassium persulphate - 0.05-0.1 sodium metabisulphate - 0.01 -0.2.
The above initiating system makes it possible to receive a copolymer of acrylamide and acrylamide with a more regular structure, which ensures better physical-chemical characteristics.
The method of obtaining a solid nutrient medium for cultivating microorganisms is carried out as follows.
First, the monomers of acrylamide and N,N'-methylene-bis-acrylamide are rid of all traces of acrylic acid. The acrylamide used: C3H5NO; molecular weight:
71.08; white odorless crystaline powder; melting temperature: 84.5+0.3C.
Density: 1.122 g/cm3; Solubility: 215.5 per 100 9 water at 25C; also dissolves in methanol, ethanol, acetone, chloroform and benzol.
It is possible to use acrylamide manufactured by REANAL (Hungary) or ALDRICH (U.S.), where the main agent is 98.6% and acrylic acid, 0.02%; also acrylamide made by ELECTRAN (U.K.), where the main agent is 99.9%, acrylic acid 0.02%. The N,N'-methylene-bis-acrylamide used: C7H,0N203; mol. weight 154.16;
white odorless crystaline powder; melting temperature 185C; solubility 39 per 100 g water at 20C. It is also possible to use N,N'-methylene-bis-acrylamide manufactured by REANOL (Hungary), FLUKA-CHEMIKA (Switzerland), where the main agent is 96.8%, acrylic acid 0.02%, or that made by ELECTRON (U.K.), where the main agent is 99.9%, acrylic acid 0.02%.
The aforementioned monomers can be purified of all traces of acrylic acid, for example, by recrystalization.
The recrystalization of acrylamide is carried out as follows: 709 acrylamide is dissolved in 1 liter of chloroform at 50-60C, then filtered while still hot. The filtrate is cooled in a refrigerator till -15 to -20C. The fallout crystals are filtered and rinsed on the filter with cold chloroform. After drying out their melting temperature is determined.
The content of the main agent increases up to 99.0% when ~A21 5~720 using acrylamide made by REANOL (Hungary) or ALDRICH (U.S.), with no traces of acrylic acid. If ELECTRON (U.K.) acrylamide is applied, the content of the main agent rises up to 99.5%, with no traces of acrylic acid.
N,N'-methylene-bis-acrylamide is recrystalized in acetone: 36 g of N,N'-methylene-bis-acrylamide is dissolved in 1 liter of acetone, boiled with reverserefrigeration, strained through a Schott filter, cooled to a negative temperature.
The fallout crystals are filtered, too, whereupon the melting temperature is determined .
The content of the main agent increases up to 98.0% when using N,N'-methylene-bis-acrylamide made by REANAL (Hungary), FLUKA CHEMIKA
(Switzerland). If the product of ELECTRON (UK) is applied, this content rises to99.94%.
Thus purified, the monomers of acrylamide and N,N'-methylyne-bis-acrylamide can be stored for a month and be used for the preparation of solutions.
After the aforementioned purifying procedure the said monomers are used to prepare a reactive mix.
To do so, an acrylonitrile, acrylamide and N,N'methylene-bis-acrylamide are mixed in an acceptable solvent, adding an initiating system. Distilled water or sodium chloride can be used as an acceptable solvent. The components of the reactive mix have the following proportions (% by weight):
acrylonitrile - 5.0-50.0 acrylamide - 5.0-50.0 N,N'-methylene-bis-acrylamide - 0.01-0.6 initiating system - 0.015-0.03 acceptable solvent - remainder Potassium persulphate and sodium metabisulphate are used as the initiating system in the following percent-by-weight proportions:
potassium persulphate - 0.005-0.1 sodium metabisulphate - 0.01-0.2 ~A~l 50720 The reactive mix thus obtained is place in the form where the copolymerization occurs, producing a disk-shaped hydrogel. The process lasts forone hour, at 20C. The copolymer of acrylonitrile and acrylamide thus obtained is retrieved from the form, rinsed in a physiological solution and saturated. After that it is permeated with a nutrient substrate (e.g., beef-ectract broth, or using the Hottinger technique, etc., depending on the organisms to be inoculated). For example, to permeate the copolymer of acrylonitrile and acrylamide with beef-extract broth, the disk-shaped copolymer is immersed in the broth and left there for 2-3 hours at 90C. Thus produced, the solid culture media is sterilized - for example, by exposure to~ rays, by steam or in the autoclave.
The solid culture medium thus obtained contains (in % by weight):
copolymer of acrylonitrile and acrylamide - 5.0-30.0 nutrient substrate - 70.0-95.0 After drying in a thermostat at 37C, this solid culture medium is inoculated with test microorganisms like ones with varying terms of cultivation, and even - and it has been mentioned before herein - with microorganisms with prolonged terms of cultivation (e.g., microbacterias, fungi, etc.). The inoculation is carried out in accordance with conventional microbiological techniques.
Methods of saturation with other nutrient substrates will be decribed in concrete examples of the realization of the present invention.
The following are examples of concrete realization of the present invention.
Example 1.
A solid nutrient medium for cultivating microorganisms was obtained using the techniques described above herein. An acrylamid and N,N'-methylene-bis-acrylamide were used after cleansing them of acrylic acid by methods laid down above herein.
To receive a copolymer of acrylonitrile and acrylamide, 59 acrylamide, 5.0 9 acrylonitrile, 0.01 9 N,N'-methylene-bis-acrylamide, 0.005 9 potassium persulphate and 0.01 g sodium metabisulphate were mixed, adding distilled water to reach ~,`A2~ ~0720 an amount of 100 9. The resulting copolymer of acrylonitrile and acrylomide was permeated with beef-extract broth at 90C for 3 hours, then sterilized by exposure to 1 Mrad gammrays. In percent-by-weight, this solid culture medium contained:
copolymer of acrylonitrile - 5.0 and acrylamide nutrient substrate - 95.0 The solid culture medium was dried in a thermostat for 30 min. at 37C and inoculated with staphylococcus aureus. After incubation for 24 hours at 37C themedium grew typical circular golden colonies with even edges. The culture's typicality was confirmed by microbiological tests. It was also noted that the properties of the microorganisms bred in the present solid culture medium were more manifest than those from the control media. The solid culture medium thus obtained was tested for tensile strength, relative elongation, water content, Na+-K+
diffusion coefficients. The results are provided in Table 1. The latter also contains data of tests involving solid culture media as per CA 1,283,873. Table 2 provides that results of tests involving culture media as per the present invention, focused on sensitivity and effectiveness. This table also contains the results of tests of control media (e.g., plain agar and a solid culture medium as per CA 1,283,873).Example 2.
A solid culture medium was produced using the techniques described above herein. An acrylamid and N,N'-methylene-bis-acrylamide were used after cleansingthem of acrylic acid, by methods described above herein.
To receive a copolymer of acrylonitrile and acrylamide, 15 g acrylonitrile, 25 g acrylamide, 0.2 9 N,N'-methylene-bis-acrylamide, 0.08 9 potassium persulphate andO.16 9 sodium metabisulphate were mixed, adding sodium chloride solution to an amount of 100 9.
Hottinger's tryptic decoction containing 200 mg% amino nitrogen was used as the nutrient substrate. The impregnation and simultaneous sterilization of the copolymer of acrylonitrile and acrylamide was done in an autoclave. The process lasted 20 min. at 120C.
(;A21 5~72~
The solid culture medium thus obtained had these percent-by-weight components:
copolymer of acrylonitrile and acrylamide - 15.0 culture medium - 85.0 The solid culture medium was transferred to Petri dishes in aseptic conditions and dried in a thermostat for 30 min. at 37C and inoculated with Escherichia coli. After 24 hours of incubation at 37C the media showed typical E.
coli colonies - sparkling plano-convex formations measuring up to 2 mm, with even edges. The culture's typicality was confirmed by microscopic studies. It was also noted that its properties, as developed in the present solid culture medium, were more manifest than those of the microorganisms cultivated in the control medium. The results of the tests are provided in Table 1 and 2.
Example 3.
A solid culture medium was produced and acrylamide and N,N'-methylene-bis-acrylamide cleansed and administered by methods described above herein.
To receive a copolymer of acrylonitrile and acrylamide, 50 g acrylonitrile, 309 acrylamide, 0.60 9 N,N'-methylene-bis-acrylamide, 0.1 9 potassium persulphate and 0.2 9 sodium metabisulphate were mixed, adding a sodium chloridesolution to an amount of 100 9.
Sabourand's glucose agar was used as the nutrient substrate. The copolymer of acrylonitrile and acrylamide thus obtained was in the form of disks. It was impregnated for 3 hours at 90C. After that it was sterilized by exposure to1.0 Mrad ~ rays.
The ready solid culture medium had these percent-by-weight components:
copolymer of acrylonitrile - 30.0 and acrylamide nutrient substrate - 70.0 In aseptic conditions the solid culture medium was transferred to Petri dishes, dried in a thermostat at 37C and inoculated with Candida albicons. After 72 hours of incubation at 30-35C it yielded typical round creams like, whitish cupola-shaped sparkling colonies. The culture's typicality was corroborated by microscopic examination. It was also noted that its properties were more manifest than those received from the control media. The colo-~21 5~720 nies bred in the control culture media turned out dim, flat and non-typical.
The results of the tests of the present solid culture medium are given in Tab.
1 and 2.
ExamPle 4.
A solid culture medium was obtained by methods described above herein, involving an acrylamide and N,N'-methylene-bis-acrylamide purified using the techniques described above herein.
To receive a copolymer of acrylonitrile and acrylamide, 30 g acrylonitrile, 50 g acrylamide, 0.60 9 N,N'-methylene-bis-acrylamide, 0.1 9 potassium persulphate and 0.2 9 metabisulphate were mixed and water added to an amount of 100 9.
Liquid wort was used as the nutrient substrate. The ready disk-shaped copolymer of acrylonitrile and acrylamide was impregnated in an autoclave at 0.5atm., 112C. The process lasted 15 min. The solid culture medium thus obtained had these percent-by-weight components:
copolymer of acrylonitrile and acrylamide - 25.0 nutrient substrate - 75.0 In aseptic conditions the solid culture medium was transferred to Petri dishes, dried in a thermostat at 37C, and inoculated with Fusarium avenacuum.
After 336 hours of incubation at 28C it yielded typical colonies with a diameter of up to 1 cm and pink mycelium. The typicality was confirmed by microscopic examinations. Similar cultures bred in the control media yielded white myceliums, which is uncharacteristic of this culture.
The results of the tests are found in Tab. 1 and 2.
ExamPle 6 (comParative) A solid culture medium was obtained by methods described above herein.
However, the acrylamide and N,N'-methylene-bis-acrylamide used were not cleansed of the traces of acrylic acid.
To receive a copolymer of acrylonitrile and acrylamide, the amounts of acrylonitrile, acrylamide, N,N'-methylene-bis-acrylamide, potassium persulphate and metabisulphate were used as described in Example 1.
(,'A21 5~720 The solid matrix was impregnated with the nutrient medium, and the parameters of impregnation and sterilization were the same as in Example 1.
Likewise, the test culture and the process of incubation were similar to those described in Example 1. The incubation yielded smaller colonies with less pigmentation.
The results of the tests are provided in Tab. 1 and 2.
Example 6 (comparative) A solid culture medium was obtained by techniques described above herein, using an acrylamide and N,N'-methylene-bis-acrylamide that weren't previously cleansed of acrylic acid residuum.
Acrylamide, N,N'-methylene-bias-acrylamide, potassium persulphate and sodium metabisulphate were applied to receive a copolymer of acrylonitrile and acrylamide, in proportions described in Example 1.
The solid matrix was impregnated with the nutrient medium, also with parameters of impregnation and sterilization similar to those employed in Example 2. The test cultures and the process of incubation were similar to those described in Example 2.
The inoculation and incubation yielded smaller colonies (about 1 mm each) that were dim and flat.
The results of the tests are shown in Tab. 1 and 2.
Example 7. (comParative) A solid culture medium was produced by techniques described above herein, using an acrylamide and N,N'-methylene-bis-acrylamide that weren't previously cleansed of acrylic acid residuum.
Acrylamide, N,N'-methylene-bis-acrylamide, potassium persulphate and sodium metabisulphate where used to produce a copolymer of acrylonitrile and acrylamide, in proportions described in Example 3.
The impregnation of the solid matrix with the nutrient medium, sterilization, the test culture and the incubation procedures were similar to those described in Example 3.
The colonies thus obtained turned out flat and dim.
The results of the tests are provided in Tab. 1 and 2.
~A21 50720 ExamPle 8 (comParative) The same techniques were applied to produce a solid culture medium as described above herein. An acrylamide and N,N'-methylene-bis-acrylamide were used, having been cleansed of acrylic acid residuum by the present method.
To receive the solid matrix for cultivating microorganisms, 3 9 acrylamide, 1 g acrylonitrile, 0.005 9 N,N'-methylene-bis-acrylamide, 0.003 9 potassium persulphate and 0.006 9 sodium metabisulphate were mixed and a physiological salt solution added, to an amount of 100 g.
The resulting copolymer of acrylonitrile and acrylamide had the consistency of a semi-liquid gel and could not be used for the production of a solid culturemedium.
Exam~le 9 (com~arative) The same techniques were applied to produce a solid culture medium as described above herein. An acrylamide and N,N'-methylene-bis-acrylamide were used after cleansing them of all traces of acrylic acid by the present method.
To receive the solid matrix for cultivating microorganisms, 60 9 acrylamide, 309 acrylonitrile, 0.65 9 N,N'-methylene-bis-acrylamide, 0.12 9 potassium persulphate and 0.24 9 sodium metabisulphate were mixed and distilled water added, to an amount of 100 9.
The copolymer of acrylonitrile and acrylamide thus obtained turned out too fragile, with damaged structure solidity, and with air bubbles. It was therefore ill-advised to use it to produce a solid culture medium.
ExamPle 10 (comParative) The present techniques were used to produce a solid matrix for cultivating microorganisms. Acrylamide and N,N'-methylene-bis-acrylamide were used after cleansing them of acrylic acid residuum by the present method.
To receive the solid matrix, 30 9 acrylamide, 65 9 acrylonitrile, 0.65 9 N,N'-athylene-bis-acrylamide, 0.12 9 potassium persulphate and sodium metabisulphate (0.24 9) were mixed and distilled water added, to an amount of 100 9.
~21 5~720 When producing a copolymer of acrylonitrile and acrylamide, the phases dispersed and the resultant copolymer lost its clarity, so it could not be used to make a solid culture medium.
As is seen from Table 1 and Table 2, solid culture media produced by the present method show better physical-mechanical properties, as compared to those made under CA 1,283,873. Table 1 further shows that use of acrylamide and N,N'-methylene-bis-acrylamide without first cleansing them of all traces of acrylic acid (Examples 5-7), as well as use of the components in the reactive mix with parameters other than those contained in the formula of the present invention (Examples 8-10), results in the worsening of the characteristics of solid culture media. Table 2 illustrates the advantages of solid culture media prepared under the present invention, in terms of biologic indices, as compared to the control media and the data provided by Examples 5-7.
Table 1.
Indices Examples Comparative examples As per CA 1,283,873 as per this invention Tensile strength 155.0 162.0 171.0 173.0 150.0 160.0 169.0 145.0 coefficient (Pa) Relative elongation (%) 280 250 190.0 200.0 276.0 243.0 187.0 230.0 Water content (%) 95.0 85.0 70.0 75.0 95.0 86.0 70.0 93.0 Diffusion coefficient, 1 o6 cm2/seC
DNa+ 8.0 5.9 4.5 5.0 6.0 4.7 4.0 4.2 DK+ 10.1 6.7 7.0 6.1 7.9 6.0 6.3 5.9 ~ A2 ~ 50720 Table 2.
Examples I n d i c e s Sensitivity Effectiveness (billion/ml) Example 1.
As per this invention 10-7 7.0 Control Media:
10-7 6.9 plaln agar as per CA 1,283,873 10-6 5.0 Example 2.
As per this invention 10-6 6.8 Control media:
106 6.5 plaln agar as per CA 1,283,873 10-5 4.9 Example 3.
As per this invention 10-7 6.0 Control media:
10-7 5.8 plaln agar as per CA 1,283,837 10-5 4.0 Example 4.
As per this invention 10-6 6.8 Control media:
10-6 6.8 plain agar as per CA 1,283,873 10-5 4.3 Example 5 (comparative) 10-5 4.5 Example 6 (comparative) 10-4 4.1 Example 7 (comparative) 10-4 4.0
SOLID CULTURE MEDIUM
AND THE METHOD OF ITS PRODUCTION
The present invention relates to a solid nutrient medium for cultivating microorganisms and the method of its production. Solid culture media can be usedin medical practice and in biology.
There exists a method of obtaining a solid culture medium for medical and biological purposes by the copolymerization of an acrylamide and a N,N'-methylene-bis-acrylamide in a sodium chloride solution, at a ratio of accordingly 0.5-40.0: 2.5-12.0, until the formation of a polyacrilamide gel. The latter, containing a maximum amount of water, is rinsed in a sodium chloride solution and saturated with nutrient substrates (SU,- 997,466).
The culture medium thus obtained is used to study the biological properties of microorganisms. However, this culture medium, solid as it is, has a markedly vulnerable surface layer, especially in the course of sterilization, inoculation, and when removing colonies with a loop. Repeated inoculations are possible after rinsing the surface in a sodium chloride solution, which is ill-advised in most microbiological manipulations.
Another method of obtaining a solid culture medium consists in a radical copolymerization of an acrylamide and N,N'-methylene-bis-acrylamide in a sodium chloride solution at a ratio of accordingly 15.0-20.0:0.019-0.132, until the formation of a polyacrilamide gel. The gel is rinsed in a physiological solution and let saturate for 16-20 hours at 50-60C. After that the polyacrilamide gel is saturated with nutrient substrates. The solid culture medium thus produced is used for cultivating microorganisms. It is colorless, elastic and displays good adhesive properties. When inoculated, microorganisms spread over the surface, without growing into it, and are completely removed with the loop. However, during storage in Petri dishes and incubation the liquid tends to exude, the effects ofwhich are not totally liquidated even after lengthy periods of thermostatic treatment. Besides, certain types of microorganisms, particularly mobile forms, fail to show the typical growth of colonies in this culture medium despite the presence of characteristic morphological, biochemical and antigenic properties. This limitates the usage of the above culture medium, as for example, in the diagnostics of contageous diseases.
~, ~ 2 1 50720 Finally, there is the method of producing a solid culture medium using a radical polymerization of acrylamide and N,N'-methylene-bis-acrylamide at a massratio of accordingly 15.0-20.0:0.019-0.132, in a sodium chloride solution, until the formation of a polyacrilamide gel, with subsequent rinsing and saturation and permeation with nutrient substrates. Here radical polymerization takes place in the presence of polyvinyl alcohol at a weight ratio of acrylamide and polyvinyl alcohol of 15.0-20.0: 1.0-3.0 (Ca 1,283,873).
However, the structure of the polyacrilamide gel obtained using this method hinders the diffusion of the ions Na+ and K+ (that are part of most of the knownculture media) from within the polyacrilamide gel to its surface where the microorganisms are located. Consequently, the microorganisms showe poorer culture properties, especially when prolonged cultivation is required (as in the case of microbacterias, fungi, etc.).
Besides, the known solid culture medium is not sufficiently effective by its biological indices.
Here and further on we understand by "biological indices":
(a) Sensitivity which is determined by the maximum dilution of a culture, when every inoculated dish (test tube) shows a certain growth (the result reflects an appropriate dilution, with 10-'-10-9 for all the groups of culture media);
(b) Effectiveness, which is determined by the yield of microbic bodies per 1 ml of culture medium (in billion/ml).
Further, it is necessary to improve the physical-mechanical properties of solid culture media obtained using the known techniques.
The present invention is aimed at bettering the diffusive and physical-mechanical properties of a solid nutrient medium for growing mcrioorganisms, as well as at improving its biological indices.
The set goal is attained by the presence in the solid culture medium of a copolymer of acrylonitrile and acrylamide and a liquid nutrient substrate with the following ratios (% by weight):
copolymer of acrylonitrile and acrylamide - 5.0-30.0 liquid nutrient substrate- 70.0-95Ø
(,A21 5~720 The aforementioned nutrient medium for cultivating microorganisms possesses high diffusive and physical-mechanical properties, as well as significant biological indices, which ensures it a number of advantages as compared to the known solid culture media.
The set objective is also attained by the previous purification of the acrylamide and N,N'-methylene-bis-acrylamide by recrystalization as part of the present method of obtaining a solid culture medium; also by the preparation of areactive mix containing acrylonitrile, acrylamide, N,N'-methylene-bis-acrylamide, and acceptable solvent, an initiating system; by the process of polymerization over a period necessary for obtaining the copolymer retaining the form; by the rinsing of the copolymer and its saturation in water and permeation with a liquid nutrient substrate, with the following ratios of components in the reactive mix (% by weight):
acrylonitrile - 5.0 - 50.0 acrylamide - 5.0 - 50.0 methylene-bis-acrylamide - 0.01 - 0.60 initiating system - 0.015 - 0.3 acceptable solvent - the remainder The above method of obtaining a solid culture medium ensures the latter's higher diffusive properties and as a result a better diffusion of the ions Na+ and K+
(as components of most of the known solid culture media) from within the solid nutrient medium to its surface where the microorganisms are located. This, in turn, makes it possible to promote the culture properties of the microorganisms of prolonged cultivation.
Besides, the present method allows to improve the biological and physical-mechanical indices. This advantage is secured by the preparatory purification ofthe acrylamide and N,N'-methylene-bis-acrylamide by recrystalization, whereby all traces of acrylic acid are removed (and further on we show that this acid is present in all the monomers under study, to the amount of some 0.2% by weight of the agent - irrespective of the manufacturing company - and that its presence negatively affects the end product).
Finally, the positive effect of the said method is also achieved by the usage of the purified monomers acrylamide and ~, A21 5~720 N,N'-methylene-bis-acrylamide in the reactive mix of the present composition.
Potassium persulphate and sodium metabisulphate are recommended as the initiating system, in the following proportions (% by weight):
potassium persulphate - 0.05-0.1 sodium metabisulphate - 0.01 -0.2.
The above initiating system makes it possible to receive a copolymer of acrylamide and acrylamide with a more regular structure, which ensures better physical-chemical characteristics.
The method of obtaining a solid nutrient medium for cultivating microorganisms is carried out as follows.
First, the monomers of acrylamide and N,N'-methylene-bis-acrylamide are rid of all traces of acrylic acid. The acrylamide used: C3H5NO; molecular weight:
71.08; white odorless crystaline powder; melting temperature: 84.5+0.3C.
Density: 1.122 g/cm3; Solubility: 215.5 per 100 9 water at 25C; also dissolves in methanol, ethanol, acetone, chloroform and benzol.
It is possible to use acrylamide manufactured by REANAL (Hungary) or ALDRICH (U.S.), where the main agent is 98.6% and acrylic acid, 0.02%; also acrylamide made by ELECTRAN (U.K.), where the main agent is 99.9%, acrylic acid 0.02%. The N,N'-methylene-bis-acrylamide used: C7H,0N203; mol. weight 154.16;
white odorless crystaline powder; melting temperature 185C; solubility 39 per 100 g water at 20C. It is also possible to use N,N'-methylene-bis-acrylamide manufactured by REANOL (Hungary), FLUKA-CHEMIKA (Switzerland), where the main agent is 96.8%, acrylic acid 0.02%, or that made by ELECTRON (U.K.), where the main agent is 99.9%, acrylic acid 0.02%.
The aforementioned monomers can be purified of all traces of acrylic acid, for example, by recrystalization.
The recrystalization of acrylamide is carried out as follows: 709 acrylamide is dissolved in 1 liter of chloroform at 50-60C, then filtered while still hot. The filtrate is cooled in a refrigerator till -15 to -20C. The fallout crystals are filtered and rinsed on the filter with cold chloroform. After drying out their melting temperature is determined.
The content of the main agent increases up to 99.0% when ~A21 5~720 using acrylamide made by REANOL (Hungary) or ALDRICH (U.S.), with no traces of acrylic acid. If ELECTRON (U.K.) acrylamide is applied, the content of the main agent rises up to 99.5%, with no traces of acrylic acid.
N,N'-methylene-bis-acrylamide is recrystalized in acetone: 36 g of N,N'-methylene-bis-acrylamide is dissolved in 1 liter of acetone, boiled with reverserefrigeration, strained through a Schott filter, cooled to a negative temperature.
The fallout crystals are filtered, too, whereupon the melting temperature is determined .
The content of the main agent increases up to 98.0% when using N,N'-methylene-bis-acrylamide made by REANAL (Hungary), FLUKA CHEMIKA
(Switzerland). If the product of ELECTRON (UK) is applied, this content rises to99.94%.
Thus purified, the monomers of acrylamide and N,N'-methylyne-bis-acrylamide can be stored for a month and be used for the preparation of solutions.
After the aforementioned purifying procedure the said monomers are used to prepare a reactive mix.
To do so, an acrylonitrile, acrylamide and N,N'methylene-bis-acrylamide are mixed in an acceptable solvent, adding an initiating system. Distilled water or sodium chloride can be used as an acceptable solvent. The components of the reactive mix have the following proportions (% by weight):
acrylonitrile - 5.0-50.0 acrylamide - 5.0-50.0 N,N'-methylene-bis-acrylamide - 0.01-0.6 initiating system - 0.015-0.03 acceptable solvent - remainder Potassium persulphate and sodium metabisulphate are used as the initiating system in the following percent-by-weight proportions:
potassium persulphate - 0.005-0.1 sodium metabisulphate - 0.01-0.2 ~A~l 50720 The reactive mix thus obtained is place in the form where the copolymerization occurs, producing a disk-shaped hydrogel. The process lasts forone hour, at 20C. The copolymer of acrylonitrile and acrylamide thus obtained is retrieved from the form, rinsed in a physiological solution and saturated. After that it is permeated with a nutrient substrate (e.g., beef-ectract broth, or using the Hottinger technique, etc., depending on the organisms to be inoculated). For example, to permeate the copolymer of acrylonitrile and acrylamide with beef-extract broth, the disk-shaped copolymer is immersed in the broth and left there for 2-3 hours at 90C. Thus produced, the solid culture media is sterilized - for example, by exposure to~ rays, by steam or in the autoclave.
The solid culture medium thus obtained contains (in % by weight):
copolymer of acrylonitrile and acrylamide - 5.0-30.0 nutrient substrate - 70.0-95.0 After drying in a thermostat at 37C, this solid culture medium is inoculated with test microorganisms like ones with varying terms of cultivation, and even - and it has been mentioned before herein - with microorganisms with prolonged terms of cultivation (e.g., microbacterias, fungi, etc.). The inoculation is carried out in accordance with conventional microbiological techniques.
Methods of saturation with other nutrient substrates will be decribed in concrete examples of the realization of the present invention.
The following are examples of concrete realization of the present invention.
Example 1.
A solid nutrient medium for cultivating microorganisms was obtained using the techniques described above herein. An acrylamid and N,N'-methylene-bis-acrylamide were used after cleansing them of acrylic acid by methods laid down above herein.
To receive a copolymer of acrylonitrile and acrylamide, 59 acrylamide, 5.0 9 acrylonitrile, 0.01 9 N,N'-methylene-bis-acrylamide, 0.005 9 potassium persulphate and 0.01 g sodium metabisulphate were mixed, adding distilled water to reach ~,`A2~ ~0720 an amount of 100 9. The resulting copolymer of acrylonitrile and acrylomide was permeated with beef-extract broth at 90C for 3 hours, then sterilized by exposure to 1 Mrad gammrays. In percent-by-weight, this solid culture medium contained:
copolymer of acrylonitrile - 5.0 and acrylamide nutrient substrate - 95.0 The solid culture medium was dried in a thermostat for 30 min. at 37C and inoculated with staphylococcus aureus. After incubation for 24 hours at 37C themedium grew typical circular golden colonies with even edges. The culture's typicality was confirmed by microbiological tests. It was also noted that the properties of the microorganisms bred in the present solid culture medium were more manifest than those from the control media. The solid culture medium thus obtained was tested for tensile strength, relative elongation, water content, Na+-K+
diffusion coefficients. The results are provided in Table 1. The latter also contains data of tests involving solid culture media as per CA 1,283,873. Table 2 provides that results of tests involving culture media as per the present invention, focused on sensitivity and effectiveness. This table also contains the results of tests of control media (e.g., plain agar and a solid culture medium as per CA 1,283,873).Example 2.
A solid culture medium was produced using the techniques described above herein. An acrylamid and N,N'-methylene-bis-acrylamide were used after cleansingthem of acrylic acid, by methods described above herein.
To receive a copolymer of acrylonitrile and acrylamide, 15 g acrylonitrile, 25 g acrylamide, 0.2 9 N,N'-methylene-bis-acrylamide, 0.08 9 potassium persulphate andO.16 9 sodium metabisulphate were mixed, adding sodium chloride solution to an amount of 100 9.
Hottinger's tryptic decoction containing 200 mg% amino nitrogen was used as the nutrient substrate. The impregnation and simultaneous sterilization of the copolymer of acrylonitrile and acrylamide was done in an autoclave. The process lasted 20 min. at 120C.
(;A21 5~72~
The solid culture medium thus obtained had these percent-by-weight components:
copolymer of acrylonitrile and acrylamide - 15.0 culture medium - 85.0 The solid culture medium was transferred to Petri dishes in aseptic conditions and dried in a thermostat for 30 min. at 37C and inoculated with Escherichia coli. After 24 hours of incubation at 37C the media showed typical E.
coli colonies - sparkling plano-convex formations measuring up to 2 mm, with even edges. The culture's typicality was confirmed by microscopic studies. It was also noted that its properties, as developed in the present solid culture medium, were more manifest than those of the microorganisms cultivated in the control medium. The results of the tests are provided in Table 1 and 2.
Example 3.
A solid culture medium was produced and acrylamide and N,N'-methylene-bis-acrylamide cleansed and administered by methods described above herein.
To receive a copolymer of acrylonitrile and acrylamide, 50 g acrylonitrile, 309 acrylamide, 0.60 9 N,N'-methylene-bis-acrylamide, 0.1 9 potassium persulphate and 0.2 9 sodium metabisulphate were mixed, adding a sodium chloridesolution to an amount of 100 9.
Sabourand's glucose agar was used as the nutrient substrate. The copolymer of acrylonitrile and acrylamide thus obtained was in the form of disks. It was impregnated for 3 hours at 90C. After that it was sterilized by exposure to1.0 Mrad ~ rays.
The ready solid culture medium had these percent-by-weight components:
copolymer of acrylonitrile - 30.0 and acrylamide nutrient substrate - 70.0 In aseptic conditions the solid culture medium was transferred to Petri dishes, dried in a thermostat at 37C and inoculated with Candida albicons. After 72 hours of incubation at 30-35C it yielded typical round creams like, whitish cupola-shaped sparkling colonies. The culture's typicality was corroborated by microscopic examination. It was also noted that its properties were more manifest than those received from the control media. The colo-~21 5~720 nies bred in the control culture media turned out dim, flat and non-typical.
The results of the tests of the present solid culture medium are given in Tab.
1 and 2.
ExamPle 4.
A solid culture medium was obtained by methods described above herein, involving an acrylamide and N,N'-methylene-bis-acrylamide purified using the techniques described above herein.
To receive a copolymer of acrylonitrile and acrylamide, 30 g acrylonitrile, 50 g acrylamide, 0.60 9 N,N'-methylene-bis-acrylamide, 0.1 9 potassium persulphate and 0.2 9 metabisulphate were mixed and water added to an amount of 100 9.
Liquid wort was used as the nutrient substrate. The ready disk-shaped copolymer of acrylonitrile and acrylamide was impregnated in an autoclave at 0.5atm., 112C. The process lasted 15 min. The solid culture medium thus obtained had these percent-by-weight components:
copolymer of acrylonitrile and acrylamide - 25.0 nutrient substrate - 75.0 In aseptic conditions the solid culture medium was transferred to Petri dishes, dried in a thermostat at 37C, and inoculated with Fusarium avenacuum.
After 336 hours of incubation at 28C it yielded typical colonies with a diameter of up to 1 cm and pink mycelium. The typicality was confirmed by microscopic examinations. Similar cultures bred in the control media yielded white myceliums, which is uncharacteristic of this culture.
The results of the tests are found in Tab. 1 and 2.
ExamPle 6 (comParative) A solid culture medium was obtained by methods described above herein.
However, the acrylamide and N,N'-methylene-bis-acrylamide used were not cleansed of the traces of acrylic acid.
To receive a copolymer of acrylonitrile and acrylamide, the amounts of acrylonitrile, acrylamide, N,N'-methylene-bis-acrylamide, potassium persulphate and metabisulphate were used as described in Example 1.
(,'A21 5~720 The solid matrix was impregnated with the nutrient medium, and the parameters of impregnation and sterilization were the same as in Example 1.
Likewise, the test culture and the process of incubation were similar to those described in Example 1. The incubation yielded smaller colonies with less pigmentation.
The results of the tests are provided in Tab. 1 and 2.
Example 6 (comparative) A solid culture medium was obtained by techniques described above herein, using an acrylamide and N,N'-methylene-bis-acrylamide that weren't previously cleansed of acrylic acid residuum.
Acrylamide, N,N'-methylene-bias-acrylamide, potassium persulphate and sodium metabisulphate were applied to receive a copolymer of acrylonitrile and acrylamide, in proportions described in Example 1.
The solid matrix was impregnated with the nutrient medium, also with parameters of impregnation and sterilization similar to those employed in Example 2. The test cultures and the process of incubation were similar to those described in Example 2.
The inoculation and incubation yielded smaller colonies (about 1 mm each) that were dim and flat.
The results of the tests are shown in Tab. 1 and 2.
Example 7. (comParative) A solid culture medium was produced by techniques described above herein, using an acrylamide and N,N'-methylene-bis-acrylamide that weren't previously cleansed of acrylic acid residuum.
Acrylamide, N,N'-methylene-bis-acrylamide, potassium persulphate and sodium metabisulphate where used to produce a copolymer of acrylonitrile and acrylamide, in proportions described in Example 3.
The impregnation of the solid matrix with the nutrient medium, sterilization, the test culture and the incubation procedures were similar to those described in Example 3.
The colonies thus obtained turned out flat and dim.
The results of the tests are provided in Tab. 1 and 2.
~A21 50720 ExamPle 8 (comParative) The same techniques were applied to produce a solid culture medium as described above herein. An acrylamide and N,N'-methylene-bis-acrylamide were used, having been cleansed of acrylic acid residuum by the present method.
To receive the solid matrix for cultivating microorganisms, 3 9 acrylamide, 1 g acrylonitrile, 0.005 9 N,N'-methylene-bis-acrylamide, 0.003 9 potassium persulphate and 0.006 9 sodium metabisulphate were mixed and a physiological salt solution added, to an amount of 100 g.
The resulting copolymer of acrylonitrile and acrylamide had the consistency of a semi-liquid gel and could not be used for the production of a solid culturemedium.
Exam~le 9 (com~arative) The same techniques were applied to produce a solid culture medium as described above herein. An acrylamide and N,N'-methylene-bis-acrylamide were used after cleansing them of all traces of acrylic acid by the present method.
To receive the solid matrix for cultivating microorganisms, 60 9 acrylamide, 309 acrylonitrile, 0.65 9 N,N'-methylene-bis-acrylamide, 0.12 9 potassium persulphate and 0.24 9 sodium metabisulphate were mixed and distilled water added, to an amount of 100 9.
The copolymer of acrylonitrile and acrylamide thus obtained turned out too fragile, with damaged structure solidity, and with air bubbles. It was therefore ill-advised to use it to produce a solid culture medium.
ExamPle 10 (comParative) The present techniques were used to produce a solid matrix for cultivating microorganisms. Acrylamide and N,N'-methylene-bis-acrylamide were used after cleansing them of acrylic acid residuum by the present method.
To receive the solid matrix, 30 9 acrylamide, 65 9 acrylonitrile, 0.65 9 N,N'-athylene-bis-acrylamide, 0.12 9 potassium persulphate and sodium metabisulphate (0.24 9) were mixed and distilled water added, to an amount of 100 9.
~21 5~720 When producing a copolymer of acrylonitrile and acrylamide, the phases dispersed and the resultant copolymer lost its clarity, so it could not be used to make a solid culture medium.
As is seen from Table 1 and Table 2, solid culture media produced by the present method show better physical-mechanical properties, as compared to those made under CA 1,283,873. Table 1 further shows that use of acrylamide and N,N'-methylene-bis-acrylamide without first cleansing them of all traces of acrylic acid (Examples 5-7), as well as use of the components in the reactive mix with parameters other than those contained in the formula of the present invention (Examples 8-10), results in the worsening of the characteristics of solid culture media. Table 2 illustrates the advantages of solid culture media prepared under the present invention, in terms of biologic indices, as compared to the control media and the data provided by Examples 5-7.
Table 1.
Indices Examples Comparative examples As per CA 1,283,873 as per this invention Tensile strength 155.0 162.0 171.0 173.0 150.0 160.0 169.0 145.0 coefficient (Pa) Relative elongation (%) 280 250 190.0 200.0 276.0 243.0 187.0 230.0 Water content (%) 95.0 85.0 70.0 75.0 95.0 86.0 70.0 93.0 Diffusion coefficient, 1 o6 cm2/seC
DNa+ 8.0 5.9 4.5 5.0 6.0 4.7 4.0 4.2 DK+ 10.1 6.7 7.0 6.1 7.9 6.0 6.3 5.9 ~ A2 ~ 50720 Table 2.
Examples I n d i c e s Sensitivity Effectiveness (billion/ml) Example 1.
As per this invention 10-7 7.0 Control Media:
10-7 6.9 plaln agar as per CA 1,283,873 10-6 5.0 Example 2.
As per this invention 10-6 6.8 Control media:
106 6.5 plaln agar as per CA 1,283,873 10-5 4.9 Example 3.
As per this invention 10-7 6.0 Control media:
10-7 5.8 plaln agar as per CA 1,283,837 10-5 4.0 Example 4.
As per this invention 10-6 6.8 Control media:
10-6 6.8 plain agar as per CA 1,283,873 10-5 4.3 Example 5 (comparative) 10-5 4.5 Example 6 (comparative) 10-4 4.1 Example 7 (comparative) 10-4 4.0
Claims (3)
1. A solid nutrient medium for cultivating microorganisms (solid culture medium) containing a copolymer of acrylonitrile and acrylamide, and a liquid nutrient substrate with the following proportions (% by weight):
copolymer of acrylonitrile and acrylamide - 5.0-30.0 liquid nutrient substrate - 70.0-95.0
copolymer of acrylonitrile and acrylamide - 5.0-30.0 liquid nutrient substrate - 70.0-95.0
2. A method of producing a solid culture medium, as per Claim 1, including the purification of acrylamide and N,N'-methylene-bis-acrylamide by recrystalization;
preparation of a reactive mix containing acrylonitrile, acrylamide, N,N'-methylene-bis-acrylamide, an acceptable solvent and an initiating system; polymerization over a period sufficient for the production of a copolymer retaining the form; cleansing this copolymer, its saturation and impregnation with a nutrient substrate, with the following components of the reactive mix and percent-by-weight proportions:
acrylonitrile - 5.0-50.0 acrylamide - 5.0-50.0 N,N'-methylene-bis-acrylamide - 0.01-0.60 initiating system - 0.15-0.3 acceptable solvent - 0.01-02
preparation of a reactive mix containing acrylonitrile, acrylamide, N,N'-methylene-bis-acrylamide, an acceptable solvent and an initiating system; polymerization over a period sufficient for the production of a copolymer retaining the form; cleansing this copolymer, its saturation and impregnation with a nutrient substrate, with the following components of the reactive mix and percent-by-weight proportions:
acrylonitrile - 5.0-50.0 acrylamide - 5.0-50.0 N,N'-methylene-bis-acrylamide - 0.01-0.60 initiating system - 0.15-0.3 acceptable solvent - 0.01-02
3. A method, as per Claim 2, whereby the initiating system is composed of a mixture of potassium persulphate and sodium metabisulphate, with the following percent-by-weight ratios:
potassium persulphate - 0.005-0.1 sodium metabisulphate - 0.01-02
potassium persulphate - 0.005-0.1 sodium metabisulphate - 0.01-02
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU30790/92A AU3079092A (en) | 1992-12-04 | 1992-12-04 | Solid culture medium and the method of its production |
| PCT/CA1992/000530 WO1994013781A1 (en) | 1992-12-04 | 1992-12-04 | Solid culture medium and the method of its production |
| CA002150720A CA2150720A1 (en) | 1992-12-04 | 1992-12-04 | Solid culture medium and the method of its production |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CA1992/000530 WO1994013781A1 (en) | 1992-12-04 | 1992-12-04 | Solid culture medium and the method of its production |
| CA002150720A CA2150720A1 (en) | 1992-12-04 | 1992-12-04 | Solid culture medium and the method of its production |
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| CA2150720A1 true CA2150720A1 (en) | 1994-06-23 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002150720A Abandoned CA2150720A1 (en) | 1992-12-04 | 1992-12-04 | Solid culture medium and the method of its production |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2150720A1 (en) |
| WO (1) | WO1994013781A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8076026B2 (en) | 2010-02-05 | 2011-12-13 | International Battery, Inc. | Rechargeable battery using an aqueous binder |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2162198B (en) * | 1984-07-27 | 1987-12-02 | Ki Med I | Process for producing dense nutrient medium for culturing microorganisms and macroorganism cell cultures |
| JPH01503274A (en) * | 1987-05-26 | 1989-11-09 | キエフスキ メディツィンスキ インスティテュト イメニ アカデミカ アー．アー．ボゴモルツァ | Method for preparing concentrated nutrient medium for culturing microorganisms |
-
1992
- 1992-12-04 WO PCT/CA1992/000530 patent/WO1994013781A1/en active Application Filing
- 1992-12-04 CA CA002150720A patent/CA2150720A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994013781A1 (en) | 1994-06-23 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |