CA1283873C - Method of preparing dense nutrient medium for cultivating microorganisms - Google Patents
Method of preparing dense nutrient medium for cultivating microorganismsInfo
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- CA1283873C CA1283873C CA000547018A CA547018A CA1283873C CA 1283873 C CA1283873 C CA 1283873C CA 000547018 A CA000547018 A CA 000547018A CA 547018 A CA547018 A CA 547018A CA 1283873 C CA1283873 C CA 1283873C
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- acrylamide
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Abstract
Abstract A method of preparing a dense nutrient medium for cultivating microorganisms, residing in radical copoly-merization of acrylamide, N,N'-methylene-bis-acrylamide and polyvinyl alcohol at a mass ratio thereof of (15-20):
(0.019-0.132):(1.0-3.0) in a physiological salt solution up to the formation of a gel with subsequent washing and swelling of the gel in the pbysiological solution and impreg-nation of the gel with a nutrient substrate.
(0.019-0.132):(1.0-3.0) in a physiological salt solution up to the formation of a gel with subsequent washing and swelling of the gel in the pbysiological solution and impreg-nation of the gel with a nutrient substrate.
Description
The present invention relates to -the field of medicine and more particularly to a me~hod of preparing a dense nutrient medium based on synthetic gel for cultivating different types of microorganisms.
Dense nutrient media prepared by the proposed rnethod can be used for different microbiological purposes.
Known in the art is a method of preparing a dense nutrient medium for medical and biological purposes via radical copolymerization of acrylamide and N,N~-methylene-bis-acrylamide in a physiological salt solution at a mass ratio thereof of 0.5-40.0:2.5-1.20, respectively, said polymerization being performed up to the formation of a polyacrylamide gel. The gel thus obtained containing a maximum amount of water is washed with a physiological salt solu~ion and impregnated with nutrient substrates ~US, A,977466).
The prepared dense nutrient medium is used for studying the biological properties of microorganisms. SUCh a dense nutrient medium is characterized, however, by a high sensitivity towards damages of the surface layer in the course of sterilization, inoculation of microorganisms, and removal of colonies by means of a bacteriological loop. The removal of microorganisms in the course of repeated inoculations is performed by washing with a physiological salt solution, which procedure is not adopted in the majority of microbiological operations.
Also known in the art is a method of preparing a dense nutrient medium for cultivating microorgànisms, residing in that radical copolymerization of acrylamide and N,N'-methylene-bis-acrylamide is carried out in a physiological salt solution at a mass ratio thereof of 15-20:0.019-0.132, respectively up to the formation of a polyacrylamide gel. The gel thus obtained is washed with the physiological salt solu-tion and sub~ected to swelling in the physiological salt solution for 16-20 hours at 50-60Co Then the polyacrylamide gel is impregnated with nutrient substrates (DE, A, 3~30316~2~
The resulting ~ense nutrient rnediurn is used for cultivating microorganisms~ 'l'he medium is elastlc, transparent, and possesses high adhesion properties. The cultures of microorganisms are located on the surface of -the medium wi-thout growing into it and are completely removed by a bacteriological loop. However, exudation of the liquid is observed on the surface of this dense nutrient medium in the course of keeping thereof in Petri dishes and incubation, the exudation being observed even upon prolonged drying in an incubator. sesides, some types of microorganisms, in particular labile forms, do not give a typical growth of colonies on the above medium, while displaying specific morphological, biochemical, and antigen properties. This limits the applicability of the above-described nutrient medium, for instance, for the diagnosis of infectious diseases.
It is an object of the invention -to provide a dense nutrient medium which will ensure the growth of a wide range of microorganisms with typical biological propertles.
Said ob;ect is accomplished by that in a method of preparing a dense nutrient medium for cultivating microorganisms, the method including radical copolymerization of acrylamide and N, N'-methylene-bis-acrylamide at a mass ratio thereof of (15-20): (0~019 0~132) in a physiological salt solution to the formation of gel, washing and swelling the gel in the physiological salt solution, and impr~gnation with nutrient substrates, according to the invention, radical copolymerization is performed in the presence of polyvinyl alcohol at a mass ratio of acrylamide and polyvinyl alcohol of 15-20 1~0-3~0~
Polyvinyl alcohol can be used as a aqueous solution.
'~' ..~
Solutions or acrylamide, N,N'-methylene-bis-acrylamide, initiators, and polyvinyl alcohol in a physiological salt solution containing 0.8-0.9 mass ~ of sodium chloride are prepared. As initiators use is made of N,N'-tetramethylethylenediamine and ammonium persulphate.
The reaction mix-ture containing acrylamide, N,N'-methylene-bis-acrylamide, and polyvinyl alcohol in a mass ratlo of (15:20):(0.019-0.132):(1.0-30) is prepared from the solutions.
The reaction mixture is supplied to a reactor and radical copolymerization is performed to the formation of a polyacrylamide gel in the form of discs. Gel sys-tems with a high moisture content are known to be suEficiently labile. A polymer-liguid phase equilibrium attained in the course of gel preparation is disturbed under the effect of various factors, for instance, temperature and humidity. Therefore, it is required to stabilizP the polyacrylamide gel obtained by effecting an additional structurization of the gel in the course o~
preparation thereof. For this purpose polyvinyl alcohol is introduced into the reaction mixture, preferably in the form of an aqueous solution for a better distribution within the mass of the reaction mi*ture. The solution of polyvinyl alcohol in water or in the physiological salt solution is a structurized liquid.
In the course of copolymerization and gel formation an interpenetrating polymer network is formed, which stabilizes the gel system and gel retains its hydrophilic properties, which is especially important.
The amount of polyvinyl alcohol tPVA) is dictated by the stability of the substrate with time and retention of typical colony formation of labile forms of the microorganisms grown on the medium prepared on such a substrate. The introduction of a smaller amount of PVA does not stabilize the substrate sufficiently. The introduction of a greater amount of PVA
results in a loss of transparency o~ the substrate, which is .
-~3~
undesirable. For ins-tance, the amount of PVA in the reaction mixture less than 1 g does not stabilize the substrate and after incubation at 37C exuda-tion of the liquid on the surface of the substrate is observed and labile forms of microorganisms do not give typical growth of colonies, all other properties being retained.
The amount of PVA in the reaction mixture mor~ than 3 g stabilizes the substrate and ~ives a very dense substr~te, which is undesirable. The surface of the dense nutrient medium prepared on such a substrate is very dry and can be damaged by a biological loop upon inoculation. Microorganisms grown on such a medium are characterized by a nontypical growth and are poorly removable from the surface of the medium.
The gel prepared is used as a substrate for preparing a dense nutrient medium. For this purpose the gel discs are washed with a physiological salt solution and subjected to swelling 3.5-5 times in mass. This ensures the elasticity of the medium and density close to that of agarized media. Polyacrylamide gel treated in such a way is impregnated with a nutrient substrate.
As a substrate use can be made of Hottinger's tryptic digest, meat-peptone broth, and the li~e. Then the nutrient medium is sterilized, dried, and inoculated with the test-microorganism.
The prepared dense nutrient medium has a stable structure and high adhesive properties, elasticity and optimum transparency; it ensures the stability effect of the cultural properties of microorganisms of various types, in particular, of labile forms.
For a better understanding of the present invention specific examples of realizing thereof are given below by way of illustration.
Example 1 To prepare a gel, four solutions ~,B,C, and D in a physiological salt solution containing 0.85 mass % of sodium chloride are prepared by the following procedure (the amounts of components are given as calculated for 1000 ml of the solution).
1. Preparation of solution A.
5 ml of N,N'-te~ramethylethylenediamine (TMED) is dissolved in 995 ml of a physiological salt solution.
Dense nutrient media prepared by the proposed rnethod can be used for different microbiological purposes.
Known in the art is a method of preparing a dense nutrient medium for medical and biological purposes via radical copolymerization of acrylamide and N,N~-methylene-bis-acrylamide in a physiological salt solution at a mass ratio thereof of 0.5-40.0:2.5-1.20, respectively, said polymerization being performed up to the formation of a polyacrylamide gel. The gel thus obtained containing a maximum amount of water is washed with a physiological salt solu~ion and impregnated with nutrient substrates ~US, A,977466).
The prepared dense nutrient medium is used for studying the biological properties of microorganisms. SUCh a dense nutrient medium is characterized, however, by a high sensitivity towards damages of the surface layer in the course of sterilization, inoculation of microorganisms, and removal of colonies by means of a bacteriological loop. The removal of microorganisms in the course of repeated inoculations is performed by washing with a physiological salt solution, which procedure is not adopted in the majority of microbiological operations.
Also known in the art is a method of preparing a dense nutrient medium for cultivating microorgànisms, residing in that radical copolymerization of acrylamide and N,N'-methylene-bis-acrylamide is carried out in a physiological salt solution at a mass ratio thereof of 15-20:0.019-0.132, respectively up to the formation of a polyacrylamide gel. The gel thus obtained is washed with the physiological salt solu-tion and sub~ected to swelling in the physiological salt solution for 16-20 hours at 50-60Co Then the polyacrylamide gel is impregnated with nutrient substrates (DE, A, 3~30316~2~
The resulting ~ense nutrient rnediurn is used for cultivating microorganisms~ 'l'he medium is elastlc, transparent, and possesses high adhesion properties. The cultures of microorganisms are located on the surface of -the medium wi-thout growing into it and are completely removed by a bacteriological loop. However, exudation of the liquid is observed on the surface of this dense nutrient medium in the course of keeping thereof in Petri dishes and incubation, the exudation being observed even upon prolonged drying in an incubator. sesides, some types of microorganisms, in particular labile forms, do not give a typical growth of colonies on the above medium, while displaying specific morphological, biochemical, and antigen properties. This limits the applicability of the above-described nutrient medium, for instance, for the diagnosis of infectious diseases.
It is an object of the invention -to provide a dense nutrient medium which will ensure the growth of a wide range of microorganisms with typical biological propertles.
Said ob;ect is accomplished by that in a method of preparing a dense nutrient medium for cultivating microorganisms, the method including radical copolymerization of acrylamide and N, N'-methylene-bis-acrylamide at a mass ratio thereof of (15-20): (0~019 0~132) in a physiological salt solution to the formation of gel, washing and swelling the gel in the physiological salt solution, and impr~gnation with nutrient substrates, according to the invention, radical copolymerization is performed in the presence of polyvinyl alcohol at a mass ratio of acrylamide and polyvinyl alcohol of 15-20 1~0-3~0~
Polyvinyl alcohol can be used as a aqueous solution.
'~' ..~
Solutions or acrylamide, N,N'-methylene-bis-acrylamide, initiators, and polyvinyl alcohol in a physiological salt solution containing 0.8-0.9 mass ~ of sodium chloride are prepared. As initiators use is made of N,N'-tetramethylethylenediamine and ammonium persulphate.
The reaction mix-ture containing acrylamide, N,N'-methylene-bis-acrylamide, and polyvinyl alcohol in a mass ratlo of (15:20):(0.019-0.132):(1.0-30) is prepared from the solutions.
The reaction mixture is supplied to a reactor and radical copolymerization is performed to the formation of a polyacrylamide gel in the form of discs. Gel sys-tems with a high moisture content are known to be suEficiently labile. A polymer-liguid phase equilibrium attained in the course of gel preparation is disturbed under the effect of various factors, for instance, temperature and humidity. Therefore, it is required to stabilizP the polyacrylamide gel obtained by effecting an additional structurization of the gel in the course o~
preparation thereof. For this purpose polyvinyl alcohol is introduced into the reaction mixture, preferably in the form of an aqueous solution for a better distribution within the mass of the reaction mi*ture. The solution of polyvinyl alcohol in water or in the physiological salt solution is a structurized liquid.
In the course of copolymerization and gel formation an interpenetrating polymer network is formed, which stabilizes the gel system and gel retains its hydrophilic properties, which is especially important.
The amount of polyvinyl alcohol tPVA) is dictated by the stability of the substrate with time and retention of typical colony formation of labile forms of the microorganisms grown on the medium prepared on such a substrate. The introduction of a smaller amount of PVA does not stabilize the substrate sufficiently. The introduction of a greater amount of PVA
results in a loss of transparency o~ the substrate, which is .
-~3~
undesirable. For ins-tance, the amount of PVA in the reaction mixture less than 1 g does not stabilize the substrate and after incubation at 37C exuda-tion of the liquid on the surface of the substrate is observed and labile forms of microorganisms do not give typical growth of colonies, all other properties being retained.
The amount of PVA in the reaction mixture mor~ than 3 g stabilizes the substrate and ~ives a very dense substr~te, which is undesirable. The surface of the dense nutrient medium prepared on such a substrate is very dry and can be damaged by a biological loop upon inoculation. Microorganisms grown on such a medium are characterized by a nontypical growth and are poorly removable from the surface of the medium.
The gel prepared is used as a substrate for preparing a dense nutrient medium. For this purpose the gel discs are washed with a physiological salt solution and subjected to swelling 3.5-5 times in mass. This ensures the elasticity of the medium and density close to that of agarized media. Polyacrylamide gel treated in such a way is impregnated with a nutrient substrate.
As a substrate use can be made of Hottinger's tryptic digest, meat-peptone broth, and the li~e. Then the nutrient medium is sterilized, dried, and inoculated with the test-microorganism.
The prepared dense nutrient medium has a stable structure and high adhesive properties, elasticity and optimum transparency; it ensures the stability effect of the cultural properties of microorganisms of various types, in particular, of labile forms.
For a better understanding of the present invention specific examples of realizing thereof are given below by way of illustration.
Example 1 To prepare a gel, four solutions ~,B,C, and D in a physiological salt solution containing 0.85 mass % of sodium chloride are prepared by the following procedure (the amounts of components are given as calculated for 1000 ml of the solution).
1. Preparation of solution A.
5 ml of N,N'-te~ramethylethylenediamine (TMED) is dissolved in 995 ml of a physiological salt solution.
2. Preparation of solution B.
0.742 g of N,N'-methylene-bis-acrylamide (MBA) is dissolved in 500 ml of a physiological salt solution, heated to 60C and 470 g of acrylamide (AA) is added with stirring till complete dissolution. The solution obtained is filtered and brought up to 1000 ml with the physiological salt solution.
0.742 g of N,N'-methylene-bis-acrylamide (MBA) is dissolved in 500 ml of a physiological salt solution, heated to 60C and 470 g of acrylamide (AA) is added with stirring till complete dissolution. The solution obtained is filtered and brought up to 1000 ml with the physiological salt solution.
3. Preparation of solu-tion C.
1.78 g of ammonium persulphate is dissolved in 1000 ml of a physiological salt solution.
1.78 g of ammonium persulphate is dissolved in 1000 ml of a physiological salt solution.
4. Preparation of solution D.
100 g of polyvinyl alcohol is dissolved in 900 ml of a physiological salt solution.
The reaction mixture is prepared from the above solutions. To this end, solutions A,B,C, and D are mixed in -the following volume ratio: A:B:C:D = 16:32:52:10. The ratio of acrylamide to polyvinyl alcohol ~PVA) in the reaction mixture is 15 :1. The reaction mixture is supplied to a reac-tor where radical copolymerization takes place, yielding disc-shaped gel.
~ ~e In 60 min the gel is removed, washed with the physiological salt solution for 1 hour, and then subj~cted to swelli ng 3.5 times in mass.
The prepared gel is used as the basis for obtalning a dense nutrient medium. The discs are placed into a liquid nutrient substrate (meat-peptone broth) and kept for 2-3 hours at 56C. The obtained nu-trient medium is sterilized with steam at 120C for 30 min and after drying in a incubator at 37C is inoculated with the test-microorganism.
A dense nutrient medium possesses high adhesion properties, elasticity, density, transparency, and is not damaged by a ba~teriological loop upon inoculation of microorganisms.
Meat-peptone broth is used as a liquid nutrient substrate and golden staphylococcus serves as a test-microbe.
After the cultures are incubated at 37C for a day, typical round colonies of golden colour with even edges are grown. Microscopic investiga-tions confirm that the cultures are typical.
No exudation of the liquid is observed on the dense nutrient medium. The culture is completely removed by using a bacteriological loop.
Example 2 To obtain the reaction mixture, use is made of solutions A,B,C, and D prepared as described in Example 1. The solutions are mixed in a volume ratio A:B:C:D = 16:32:52:20 and poured into a reactor for polymeri~ation. The reaction mixture contains acrylamide and PV~ in a mass ratio of 15:2Ø In an hour the plates of the gel are treated by ~ollowing the procedure described in Example 1 up to a 5-fold increase in mass.
\
~2~ii3~73~
The prepared gel is impregna-ted wi-th a nutrien-t substrate similarly to the procedure described in Example 1.
Hottinger's tryptic digest is used as the nutrient substrate containing 200 g of amine nitrogen and Bacillus pyocyaneus serves as a test-microbe. Af-ter incubation at 37C round flat mucous colonies are grown with the formation o~ a typical blue-green soluble pigment. No exudation of the liquid is observed on the surface of the dense nutrient medium. The culture does not grow into the medium and is completely removed wi-th a bacteriological loop.
Example 3 Solutions A,C and D are used, prepared by ~ollowing the procedure described in Example 1. To prepare solution B, 0.594 g of N,N'-methylene-bis acrylamide is dissolved in 500 ml of a physiological salt solution containing 0.85 mass % of sodium chloride, 470 g of acrylamide is added, and the volume is brought up to 1000 ml with the physiological salt solution of the above concentration.
Solutions A,B,C, and D are used for preparing the reaction mixture in a volume ratio ~:B:C:D = 16:32:52:30 and copolymerization is carried out in a reactor. The reac-tion mixture contains acrylamide and polyvinyl alcohol in a mass ratio of 15:3Ø The prepared gel is treated by following the procedure described in Example 1 up to a 5-fold increase in mass.
The prepared gel is impregnated with a nutrient substrates as described in Example 1. Hottinger's digest containing 200 mg of amine nitrogen is used as the substrate.
Ciliform bacterium E. coli 055 serves as a test-microbe.
After incubation at 37C for a day, the culture is grown in the form of smooth weakly convex bright colonies with even edges. The study of the morphological and agglutlnable properties confirms that the culture is typical. No exudation of liquid is observed on the surface of the m~dium.
For the sake of comparison, cul-tures of E. coli 055 are inoculated on a dense nutrient medium prepared as described in DE,A,3430316. After incubation at 37C for a day, the culture is grown in the form of flat colonies of irregular shape. Exudation of the liquid on individual sites of the dense nutrien-t medium is observed.
Example ~
2.4 g of polyvinyl alcohol is dissolved in 250 ml of water. The solution obtained is added to a solution con-taining 0.25 g MBA, 0.~5 ml TMED, and 159 g acrylamide in 470 ml physiological salt solution con-taining 0.9 mass % of sodium chloride. The mixture of the solutions is added to a solution containing 0.35 g ammonium persulphate in 160 ml of physiological salt solution of the above concentration and placed into a reactor for polymerization.
The reaction mixture contains AA and PVA at a mass ratio of 20:3. In an hour the gel plates are removed, washed with the physiological salt solution, and kept for swelling up to a 4-fold mass increase in the physiological salt solution.
The gel thus prepared is impregnated with a nutrient substrate by following the procedure described in Example 1. As the substrate use is made of Hottinger's digest containing 200 mg of amine nitrogen. Salmonella typhimurium serves as a test microbe. After incubation at 37C smooth weakly convex colonies with even edges are grown. ~he study of the morphological, saccharolytic, and agglutinable properties confirms that the culture is typical.
.~
Example 5 0.8 g of poly~inyl alcohol is dissolved in 250 ml o~
water. The solution is added to a solu-tion containing 0.25 g MBA, 0.25 g TMED, and 159 g acrylamide in ~70 ml of a physiological salt solution containing 0.8 mass % of sodium chloride. The mixture obtained is added to a solution containing 0.35 g of ammonium persulphate in 160 ml of physiological salt solution and poured into a reactor for polymerization.
The reaction mixture contains acrylamide and polyvinyl alcohol at a mass ratio of 20:1Ø In an hour the gel plates are washed with the physiological salt solution and sub;ected to swelling up to a 5.5-fold increase in mass in the physiological salt solution.
The gel obtained is saturated with the nutrient substrate by following the procedure described in Example 1. As the substrate used is made of Hottinger's digest containing 200 mg of amine nitrogen and 2~ of glucose. Strepococcus pyogenes serves as a test culture which grows as typical colonies.
Example 6 ~0 Solutions A,C, and D prepared as described in Example 1 are used. To prepare solution B, 4.125 g of N,N'-methylene-bis-acrylamide is dissolved in 500 ml of a physiological salt solution containing 0.9 mass % of sodium chloride. Then 470 g of acrylamide is added and the volume is brought up to 1000 ml with the physiological salt solution of said concentration. From all the solutions a mixture is prepared (A:B:C:D = 16:32:52:15) and copolymerization is carried out in a reactor. In the reaction mixture the mass ratio o~ acrylamide to N,N'-methylene-bis-acrylamide is 15:0.132 and o~ acrylamide to polyvinyl alcohol is 15:1.5.
~ .~
The gel obtained is trea-ted by ~ollowing the procedure described in Example 1 and the mass of -the gel is increased 3.5 times. The gel is sa-turated with a nutrient subs-tra-te as described in Example l. Meat-peptone bro-th is used as khe substrate. Shigella Flexneri cul-ture serves as a test-microbe.
After a day incuba-tion in an incubator at 37C the culture is grown as bri~ht weakly convex colonies wi-th even edges. The study of the morphological, saccharolytic, and agglutinable properties confirms that the culture is typical. No exuda-tion of liquid is observed on the surface of the medium.
Example 7 Solutions A,C, and D are used, prepared by following the procedure described in Example 1. To prepare solution s, 0.594 g of N,N'-methylene-bis-acrylamide is dissolved in 500 ml of a physiological salt solution containing 0.85 mass % of sodium chloride. Then 25 g of acrylamide is added and the volume is brought up to 1000 ml with the physiological salt solution. The above solutions in a volume ratio A:B:C:D = 16:32:52:30 are used for preparing a mixture an~ copolymerization is carried out in a reactor. In the reaction mixture the mass ratio of acrylamide and N,N'-methylene-bis-acrylamide is 20:0.019.
The gel obtained is treated as described in Example 1 to a 4.6-fold increase in mass. The gel is impregnated with a nutrient substrate by following the procedure described in Example 1. Tryptic Hottinger's digest containing 300 mg of amine nitrogen is used as the nutrient substrate. Proteus vulgaris serves as a test--microbe. After incubation for ~4 hours the culture is grown as typical moderately bright weakly convex colonies of irregular shape. The study of the morphological properties con~irms that the culture is typical. No exudation of liquid on the surface of the nutrient medium is observed.
~3~3 Examp:Le 8 Solutions A,C, and D prepared by following the procedur0 described in Example 1 are used. To prepare solu-tion B, ~.135 g o~ N,N~-methyl-bis-acrylamide is dissolved in 500 ml of a physiological salt solution containing 0.8 rnass ~ of sodium chloride. Then 25 g of acrylamide is added and the volume is brought up to 1000 ml with the physiological salt solution of said concentration. All -the above solutions are used for preparing a mixture (A:B:C:D =16:32:52:10) and copolymerization is carried out in a reactor. The mass ratio of acrylamide and polyvinyl alcohol in the reaction mixture is 20:1Ø
The obtained gel is -treated by following the procedure described in Example 1 up to a ~.0-fold increase in mass. The gel is impregnated with the nutrient substrate by following the procedurs described in Example 1. Meat-peptone broth is used as the substrate. Coliform bacterium E. coli 0111 serves as a tes-t-culture. After incubation at 37C the culture is grown as bright round weakly convex colonies with even edges and smooth surface.
The study of the morphological, saccharolytic and aggulatinable properties confirms that the culture is typical. No exudation of liquid is observed on the surface of the nutrient medium.
Example 9 Solutions A,C, and D prepared by following the procedure described in Example 1 are used. To prepare solution B, 1.78 g of N,N'-methylene-bis-acrylamide is dissolved in 500 ml of a physiological salt solution containing 0.85 mass % of sodium chloride. Then 625 g of acrylamide i5 added and the volume is brought up to 1000 ml with the physiological salt solution o said concentration. The above solutions in a ratio A:B:C:D=16032:53:25 are used for prepariny a mixture and copolymerization is carried out in a reactor. The mass ratio o~
acrylamide and N,N'-methylerle-bis-acrylamide in the reaction mixture is 20:0.057 and that oE acrylamide and polyvinyl alcohol is 20:2.5.
The obtained gel is treated by following the procedure described in Example 1 up to a 4.3-fold increase in mass. The gel is irnpregnated with a nutrient substrate by following the procedure described in Example 1. Difco broth is used as the substrate and Bac. cereus serves as a test-microbe. After incubation for 24 hours in an lncubator the culture ls grown as typical round flat colonies wi-th a rough surface. The morphological study confirms that the culture is typical. No exudation of liquid is observed on the surface of the nutrient medium.
. ~ ~
.,
100 g of polyvinyl alcohol is dissolved in 900 ml of a physiological salt solution.
The reaction mixture is prepared from the above solutions. To this end, solutions A,B,C, and D are mixed in -the following volume ratio: A:B:C:D = 16:32:52:10. The ratio of acrylamide to polyvinyl alcohol ~PVA) in the reaction mixture is 15 :1. The reaction mixture is supplied to a reac-tor where radical copolymerization takes place, yielding disc-shaped gel.
~ ~e In 60 min the gel is removed, washed with the physiological salt solution for 1 hour, and then subj~cted to swelli ng 3.5 times in mass.
The prepared gel is used as the basis for obtalning a dense nutrient medium. The discs are placed into a liquid nutrient substrate (meat-peptone broth) and kept for 2-3 hours at 56C. The obtained nu-trient medium is sterilized with steam at 120C for 30 min and after drying in a incubator at 37C is inoculated with the test-microorganism.
A dense nutrient medium possesses high adhesion properties, elasticity, density, transparency, and is not damaged by a ba~teriological loop upon inoculation of microorganisms.
Meat-peptone broth is used as a liquid nutrient substrate and golden staphylococcus serves as a test-microbe.
After the cultures are incubated at 37C for a day, typical round colonies of golden colour with even edges are grown. Microscopic investiga-tions confirm that the cultures are typical.
No exudation of the liquid is observed on the dense nutrient medium. The culture is completely removed by using a bacteriological loop.
Example 2 To obtain the reaction mixture, use is made of solutions A,B,C, and D prepared as described in Example 1. The solutions are mixed in a volume ratio A:B:C:D = 16:32:52:20 and poured into a reactor for polymeri~ation. The reaction mixture contains acrylamide and PV~ in a mass ratio of 15:2Ø In an hour the plates of the gel are treated by ~ollowing the procedure described in Example 1 up to a 5-fold increase in mass.
\
~2~ii3~73~
The prepared gel is impregna-ted wi-th a nutrien-t substrate similarly to the procedure described in Example 1.
Hottinger's tryptic digest is used as the nutrient substrate containing 200 g of amine nitrogen and Bacillus pyocyaneus serves as a test-microbe. Af-ter incubation at 37C round flat mucous colonies are grown with the formation o~ a typical blue-green soluble pigment. No exudation of the liquid is observed on the surface of the dense nutrient medium. The culture does not grow into the medium and is completely removed wi-th a bacteriological loop.
Example 3 Solutions A,C and D are used, prepared by ~ollowing the procedure described in Example 1. To prepare solution B, 0.594 g of N,N'-methylene-bis acrylamide is dissolved in 500 ml of a physiological salt solution containing 0.85 mass % of sodium chloride, 470 g of acrylamide is added, and the volume is brought up to 1000 ml with the physiological salt solution of the above concentration.
Solutions A,B,C, and D are used for preparing the reaction mixture in a volume ratio ~:B:C:D = 16:32:52:30 and copolymerization is carried out in a reactor. The reac-tion mixture contains acrylamide and polyvinyl alcohol in a mass ratio of 15:3Ø The prepared gel is treated by following the procedure described in Example 1 up to a 5-fold increase in mass.
The prepared gel is impregnated with a nutrient substrates as described in Example 1. Hottinger's digest containing 200 mg of amine nitrogen is used as the substrate.
Ciliform bacterium E. coli 055 serves as a test-microbe.
After incubation at 37C for a day, the culture is grown in the form of smooth weakly convex bright colonies with even edges. The study of the morphological and agglutlnable properties confirms that the culture is typical. No exudation of liquid is observed on the surface of the m~dium.
For the sake of comparison, cul-tures of E. coli 055 are inoculated on a dense nutrient medium prepared as described in DE,A,3430316. After incubation at 37C for a day, the culture is grown in the form of flat colonies of irregular shape. Exudation of the liquid on individual sites of the dense nutrien-t medium is observed.
Example ~
2.4 g of polyvinyl alcohol is dissolved in 250 ml of water. The solution obtained is added to a solution con-taining 0.25 g MBA, 0.~5 ml TMED, and 159 g acrylamide in 470 ml physiological salt solution con-taining 0.9 mass % of sodium chloride. The mixture of the solutions is added to a solution containing 0.35 g ammonium persulphate in 160 ml of physiological salt solution of the above concentration and placed into a reactor for polymerization.
The reaction mixture contains AA and PVA at a mass ratio of 20:3. In an hour the gel plates are removed, washed with the physiological salt solution, and kept for swelling up to a 4-fold mass increase in the physiological salt solution.
The gel thus prepared is impregnated with a nutrient substrate by following the procedure described in Example 1. As the substrate use is made of Hottinger's digest containing 200 mg of amine nitrogen. Salmonella typhimurium serves as a test microbe. After incubation at 37C smooth weakly convex colonies with even edges are grown. ~he study of the morphological, saccharolytic, and agglutinable properties confirms that the culture is typical.
.~
Example 5 0.8 g of poly~inyl alcohol is dissolved in 250 ml o~
water. The solution is added to a solu-tion containing 0.25 g MBA, 0.25 g TMED, and 159 g acrylamide in ~70 ml of a physiological salt solution containing 0.8 mass % of sodium chloride. The mixture obtained is added to a solution containing 0.35 g of ammonium persulphate in 160 ml of physiological salt solution and poured into a reactor for polymerization.
The reaction mixture contains acrylamide and polyvinyl alcohol at a mass ratio of 20:1Ø In an hour the gel plates are washed with the physiological salt solution and sub;ected to swelling up to a 5.5-fold increase in mass in the physiological salt solution.
The gel obtained is saturated with the nutrient substrate by following the procedure described in Example 1. As the substrate used is made of Hottinger's digest containing 200 mg of amine nitrogen and 2~ of glucose. Strepococcus pyogenes serves as a test culture which grows as typical colonies.
Example 6 ~0 Solutions A,C, and D prepared as described in Example 1 are used. To prepare solution B, 4.125 g of N,N'-methylene-bis-acrylamide is dissolved in 500 ml of a physiological salt solution containing 0.9 mass % of sodium chloride. Then 470 g of acrylamide is added and the volume is brought up to 1000 ml with the physiological salt solution of said concentration. From all the solutions a mixture is prepared (A:B:C:D = 16:32:52:15) and copolymerization is carried out in a reactor. In the reaction mixture the mass ratio o~ acrylamide to N,N'-methylene-bis-acrylamide is 15:0.132 and o~ acrylamide to polyvinyl alcohol is 15:1.5.
~ .~
The gel obtained is trea-ted by ~ollowing the procedure described in Example 1 and the mass of -the gel is increased 3.5 times. The gel is sa-turated with a nutrient subs-tra-te as described in Example l. Meat-peptone bro-th is used as khe substrate. Shigella Flexneri cul-ture serves as a test-microbe.
After a day incuba-tion in an incubator at 37C the culture is grown as bri~ht weakly convex colonies wi-th even edges. The study of the morphological, saccharolytic, and agglutinable properties confirms that the culture is typical. No exuda-tion of liquid is observed on the surface of the medium.
Example 7 Solutions A,C, and D are used, prepared by following the procedure described in Example 1. To prepare solution s, 0.594 g of N,N'-methylene-bis-acrylamide is dissolved in 500 ml of a physiological salt solution containing 0.85 mass % of sodium chloride. Then 25 g of acrylamide is added and the volume is brought up to 1000 ml with the physiological salt solution. The above solutions in a volume ratio A:B:C:D = 16:32:52:30 are used for preparing a mixture an~ copolymerization is carried out in a reactor. In the reaction mixture the mass ratio of acrylamide and N,N'-methylene-bis-acrylamide is 20:0.019.
The gel obtained is treated as described in Example 1 to a 4.6-fold increase in mass. The gel is impregnated with a nutrient substrate by following the procedure described in Example 1. Tryptic Hottinger's digest containing 300 mg of amine nitrogen is used as the nutrient substrate. Proteus vulgaris serves as a test--microbe. After incubation for ~4 hours the culture is grown as typical moderately bright weakly convex colonies of irregular shape. The study of the morphological properties con~irms that the culture is typical. No exudation of liquid on the surface of the nutrient medium is observed.
~3~3 Examp:Le 8 Solutions A,C, and D prepared by following the procedur0 described in Example 1 are used. To prepare solu-tion B, ~.135 g o~ N,N~-methyl-bis-acrylamide is dissolved in 500 ml of a physiological salt solution containing 0.8 rnass ~ of sodium chloride. Then 25 g of acrylamide is added and the volume is brought up to 1000 ml with the physiological salt solution of said concentration. All -the above solutions are used for preparing a mixture (A:B:C:D =16:32:52:10) and copolymerization is carried out in a reactor. The mass ratio of acrylamide and polyvinyl alcohol in the reaction mixture is 20:1Ø
The obtained gel is -treated by following the procedure described in Example 1 up to a ~.0-fold increase in mass. The gel is impregnated with the nutrient substrate by following the procedurs described in Example 1. Meat-peptone broth is used as the substrate. Coliform bacterium E. coli 0111 serves as a tes-t-culture. After incubation at 37C the culture is grown as bright round weakly convex colonies with even edges and smooth surface.
The study of the morphological, saccharolytic and aggulatinable properties confirms that the culture is typical. No exudation of liquid is observed on the surface of the nutrient medium.
Example 9 Solutions A,C, and D prepared by following the procedure described in Example 1 are used. To prepare solution B, 1.78 g of N,N'-methylene-bis-acrylamide is dissolved in 500 ml of a physiological salt solution containing 0.85 mass % of sodium chloride. Then 625 g of acrylamide i5 added and the volume is brought up to 1000 ml with the physiological salt solution o said concentration. The above solutions in a ratio A:B:C:D=16032:53:25 are used for prepariny a mixture and copolymerization is carried out in a reactor. The mass ratio o~
acrylamide and N,N'-methylerle-bis-acrylamide in the reaction mixture is 20:0.057 and that oE acrylamide and polyvinyl alcohol is 20:2.5.
The obtained gel is treated by following the procedure described in Example 1 up to a 4.3-fold increase in mass. The gel is irnpregnated with a nutrient substrate by following the procedure described in Example 1. Difco broth is used as the substrate and Bac. cereus serves as a test-microbe. After incubation for 24 hours in an lncubator the culture ls grown as typical round flat colonies wi-th a rough surface. The morphological study confirms that the culture is typical. No exudation of liquid is observed on the surface of the nutrient medium.
. ~ ~
.,
Claims (10)
1. In a method of preparing a dense nutrient medium for cultivating micro oraganisms, comprising radical copolymerization of acrylamide and N, N'-methylene, bis-acrylamide at a mass ratio thereof of from about 15-20: 0.019-0.132 in a physiological salt solution up to the formation of a gel, washing and swelling of the gel in the physiological salt solution and impregnation of the gel with a nutrient substrate, the improvement which comprises carrying out the colpolymerization in the presence of polyvinyl alcohol at a mass ratio of acrylamide and polyvinyl alcohol of from about 15-20:
about 1.0 - 3Ø
about 1.0 - 3Ø
2. The method as claimed in claim 1, wherein polyvinyl alcohol is present as an aqueous solution.
3. The method as claimed in claim 1, wherein the nutrient substrate is selected from the group consisting of Hottinger's tryptic digest, meat-peptone broth and DifCo broth.
4. The method of claim 1 or Z, wherein the nutrient substrate is Hottinger's tryptic digest.
5. The method of claim 1 or 2, wherein the nutrient substrate is meat-peptone broth.
6. The method of claim 1 or 2, wherein the nutrient substrate is Difco broth.
7. The dense nutrient medium prepared in accordance with the method of claim 1.
8. The dense nutrient medium of claim 7, which is in the form of a disco
9. In a method for cultivating a micro organism the improvement which comprises using as the cultivating medium the dense nutrient medium prepared by the method of claim 3.
10. A method according to claim 9, in which the nutrient used is in the form of a disc.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000547018A CA1283873C (en) | 1987-09-16 | 1987-09-16 | Method of preparing dense nutrient medium for cultivating microorganisms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000547018A CA1283873C (en) | 1987-09-16 | 1987-09-16 | Method of preparing dense nutrient medium for cultivating microorganisms |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1283873C true CA1283873C (en) | 1991-05-07 |
Family
ID=4136457
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000547018A Expired - Fee Related CA1283873C (en) | 1987-09-16 | 1987-09-16 | Method of preparing dense nutrient medium for cultivating microorganisms |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1283873C (en) |
-
1987
- 1987-09-16 CA CA000547018A patent/CA1283873C/en not_active Expired - Fee Related
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