GB2162198A - Process for producing dense nutrient medium for culturing microorganisms and macroorganism cell cultures - Google Patents

Abstract

A process for producing a dense nutrient medium for culturing a microorganism and cell cultures of a macroorganism wherein copolymerization of acrylamide and N,N'-methylene-bis-acrylamide is conducted at a weight ratio thereof equal to 15.0-20.0:0.19-0.132 respectively and the polyacrylamide gel resulting from the copolymerization and washing is kept in a physiological solution till its mass is increased by 2.8-4.5 times, followed by treatment thereof with a liquid nutrient medium, or the polyacrylamide gel after washing is subjected to swelling in a liquid nutrient medium till its mass is increased by 2.5-3.5 times. The present invention is especially useful in medicine and biology.

Description

SPECIFICATION Process for producing dense nutrient medium for culturing microorganisms and macroorganism cell cultures The present invention relates to medicine and biology and, more specifically, to a process for producing a nutrient medium for culturing microorganisms and macroorganisms cell cultures.

At the present time widely employed in the microbiological practice are dense nutrient media based on a naturally-occuring gel -agar-agar. However, this natural material is rather expensive and rarely available. For this reason, it is an urgent problem in the art to develop nutrient media based on synthetic gels which could substitute agar-agar in the microbiological practice.

Known in the art is a process for producing a nutrient medium for cultivation of microorganisms comprising copolymerization of acrylamide and N,N'-methylene-bis-acrylamide in an aqueous medium, followed by water washing of the resulting polyacrylamide gel to remove toxic initial monomers, impregnation thereof with a nutrient substrate, followed by sterilization (of. USSR Inventor's Certificate No.659619).

The main advantages resulting from the use of a polyacrylamide gel as a base for a nutrient medium result in that it has a permanent composition, contains no foreign matter and substances hindering growth of microorganisms after water washing, it is a highly transparent material and necessitates no clarification before using. However, the polyacrylamide gel employed in the prior art process as a dense base for a nutrient medium features an increased brittleness due to a high degree of cross-linking of polyacrylamide which hinders diffusion of high-molecular fractions of the nutrient substrate. The removal of microorganisms grown on such nutrient media is effected only by washing-off with a physiological solution.

Also known in the art is a polyacrylamide gel for biological and medical purposes and a process for making same (of. USSR Inventor's Certificate No.977466). The polyacrylamide gel according to this reference can be also used as a base for a dense nutrient medium. It is prepared by copolymerization of acrylamide and N,N'-methylene-bis-acrylamide taken in a weight ratio of 0.5-40.0:2.5-12.0 in a 0.5% sodium chloride solution in the presence of a radical-type initiator.

The resulting polyacrylamide gel which, already after the copolymerization process, contains a maximum possible amount of water is washed with a 0.5% aqueous solution of sodium chloride for 12 hours, replacing the solution every 4 hours to remove the unreacted monomers. This polacrylamide gel contains, per cent by mass: polyacrylamide 3.0 - 28.0 0.5% sodium chloride solution 72.0 - 97.0 Then the washed gel is saturated with substrates for nutrition of microorganisms, for example with meat-peptone broth.

The dense nutrient medium produced by the process described in the USSR Inventor's Certificate referred to contains no toxic monomers and can be used for the study of biological properties of microorganisms, human and animal cells.

The above-described dense nutrient medium has a high sensitivity of the superficial layer of the polyacrylamide gel to damages due to brittleness thereof upon various handling operations therewith, for example during sterlization, in inoculation of microorganisms and in the withdrawal of colonies by means of a bacteriological loop. The removal of microorganisms upon reinoculations is possible only by washing off with a physiological solution which is inacceptable in the majority of microbilogical operations. The work becomes especially complicated upon a long-time culturing of microorganisms.

It is an object of the present invention to provide such a process for producing a dense nutrient medium for growing microorganisms and celle cultures which would impart improved physico-mechanical characteristics to this dense nutrient medium enlarging the range of its utilization for microbiological purposes.

This object is accomplished by a process for producing a dense nutrient medium for culturing of microorganisms and cell cultures of a macroorganism comprising copolymerization of acrylamide and methylene-bis-acrylamide in a physiological solution in the presence of a radical-type initiator till the formation of a polyacrylamide gel, followed by washing thereof with a physiological solution, wherein according to the present invention, the copolymerization of acrylamide and N,N'-methylene- bis-acrylamide is carried out at a weight ratio thereof equal to 15.0- 20.0::0.019-0.132 respectively and the resulting, after washing, polyacrylamide gel is kept in the physiological solution till its mass is increased by 2.8-4.5 times, followed by treatment thereof with a liquid nutrient medium, or the polyacrylamide gel resulting from washing is subjected to swelling in a liquid nutrient medium till its mass is increased by 2.5-3.5 times.

The dense nutrient medium produced by the process according to the present invention is resistant to various factors and is not damaged upon handling thereof in various operations; its density is close to that of agar-agar. It is flexible, has good adhesive properties (well adheres to Petri dishes). In contrast to a known dense nutrient medium, the dense nutrient medium produced according to the present invention ensures a good sliding of a bacteriological loop upon inoculation and removal of microorganisms without damaging its surface.

Cultures of microorganisms are located on the surface of the dense nutrient medium without growing into it, thus ensuring optimal conditions for vital activity of microorganisms, whereby the biomass yield is increased. To improve quality of the dense nutrient medium, it is advisable to carry out the copolymerization at a weight ratio of the mixture of acrylamide and N,N'-methylene-bis-acrylamide to the physiological solution equal to 1:4-7 respectively. This ratio of the components in the polymerization mixture provides optimal conditions for the formation of such a spatial structure of the formed polyacrylamide gel which ensures good physico-mechanical properties of the nutrient medium prepared therefrom.

To enlarge the scope of application of the dense nutrient medium according to the present invention for the microbiological purposes, it is advisable to use, as the physiological solution, a 0.85% aqueous solution of sodium chloride or Ringer-Lock solution, Earl solution, Hanks solution of a 5% aqueous solution of glucose, while as the liquid nutrient medium it is preferable to use Hottinger tryptic overcook, Marten broth, meat-peptone broth, peptone water, casein hydrolyzate, wort, medium 199.

For cultivation of gonococci, it is advisable that the polyacrylamide gel swollen in the physiological solution be treated with a liquid nutrient medium comprising a mixture of inactivated sterile human blood serum and plasmol taken in the weight ratio of 1:1. This dense nutrient medium ensures a high quality of growth of the culture of gonococci with preservation of their morphological tinctorial, cultural and other biological properties. The use of this medium will enlarge diagnosis opportunities of bacteriology in revealing gonorrhea, especially in its chronical and torpid forms.The time required for the preparation of this dense nutrient medium is reduced by several times as compared to that required for the preparation of an ascitic meat-peptone agar, Baily agar, serum agar employed for culturing of gonococci; it also precludes the necessity in using expensive components for its preparation.

To enlarge the scope of application of the dense nutrient medium according to the present invention, it is advisable that the polyacrylamide gel be shaped into two flat plates, one which has recesses positioned coaxially and/or in a stagger-like manner, whereafter the plates are impregnated with a solution containing acrylamide and N,N'- methylene-bis-acrylamide at a weight ratio thereof of 15.0-20.0:0.0190.132 and adjust them so as to form closed cavities for a subsequent culturing, therein, microorganisms or macroorganisms cell cultures, the plates are then kept till the formation of a single block, followed by division thereof into parts, each having at least one of those closed cavities, swelling in a physilogical solution and treatment in a liquid nutrient medium.

The resulting parts with closed cavities can be referred to as diffusion chambers which have a good strength, elasticity, thermal stability owing to the polyacrylamide gel properties. They are readily reproducible, can be stored for a long time and subjected to sterilization. This dense nutrient medium shaped as diffusion chambers can be widely employed in both in vivo and in vitro systems. These diffusion chambers are atraumatic upon transplantation thereof into an organism (for example, into petiotoneal cavity of animals). They are inert for the organism which is of great importance in the study of the effect of various of the ambient medium on the objects placed into the chamber cavities.

Owing to the properties of the polyacrylamide gel, the diffusion chambers (from which they are produced) have good diffusion properties which enables easier penetration of nutrient and other substances to the studied cultures of macroorganism cells or to microorganisms placed into a chamber cavity an a greater influence on them. Furthermore, they are transparent, wherefore the test microorganisms or cell cultures are easily observed microscopically which provides an image of their organization.

The dense nutrient medium for culturing microorganisms according to the present invention is produced in the following manner First prepared is a reaction mixture containing solutions of the starting monomers in a physiological solution. The weight ratio of acrylamide to methylene- bis-acrylamide in the reaction mixture is equal to (15.0-20.0): (0.019-0.132) respectively.

As the physiological solution preferably taken in a ratio of 4-7:1 to the mixture of monomers, use is made of a 0.85% aqueous solution of sodium chloride, or a 0.9% aqueous solution of sodium chloride, or a 5% aqueous solution of glucose, or solutions of Ringer-Lock, Hanks, Earl. By varying weight proportions of the starting monomers and the physiological solution, it is possible to produce polyacrylamide gels possessing an increaded elasticity as compared to known ones which facilitates a further processing of the gel and ensures good qualities of the nutrient medium for further operations.

The copolymerization of the starting monomers can occur without heating or upon heating of the reaction mixture with the addition of known initiators. The copolymerization of the reaction mixture can be carried out in a predetermined-shape reactor such as glass, metal or ceramic vessels, as well as vessels manufactured from synthetic materials. The polyacrylamide gel produced in the copolymerization the resulting polyacrylamide gel is washed with corresponding physiological solution. Then the washed polyacrylamide gel is subjected to swelling in a physiological solution till its mass is increased by 2.8-4.5 times and the resulting gel is treated with a liquid nutrient medium comprising a tryptic overcook according to Hottinger, Marten broth, meat-peptone broth, peptone water, casein hydrolyzate, wort, medium 199.

In the second embodiment of the production of the nutrient medium according to the present invention the washed polyacrylamide gel can be subjected to swelling directly in the above-mentioned liquid nutrient medium till its mass is increased by 2.5-3.5 times. Then the swollen polyacrylamide gel is subjected to sterilization and the dense nutrient medium is ready for use. This final medium contributes to higher yields of the biomass.

The composition of liquid nutrient media is determined by the nutrition demands of particular species or groups of microorganisms or macroorganism cell cultures. For saturation and swelling of the polyacrylamide gel according to the present invention use can be made of liquid nutrient media satisfying nutrition demands of substantially all known species of microorganisms and cells including naturally-occurring, semisynethetic or synthetic compositions of substrates and mixtures thereof. The dense nutrient medium for growing microorganisms and cell cultures of macroorganisms produced by the process according to the present invention has good adhesive properties (well adheres to Petri dishes), resistant against handling operations therewith; it also ensures a good sliding of a bacterial loop upon inoculation and removal of microorganisms without damaging its surface.

Cultures of microorganisms are located on the surface of the dense nutrient according to the present invention without growing-in into it, thus providing optimal conditions for the vital activity of microorganisms and enabling higher yields of the biomass.

Since the above-mentioned properties of the dense nutrient medium of the present invention are deter mined by properties of the swollen polyacrylamide gel, it can be successfully used, as it has been found by the inventors, for the preparation of a dense nutrient medium for culturing gonococci. The polyacrylamide gel swollen in the physiological solution till its mass has become preferably 4.5 times higher is treated with a nutrient medium comprising a mixture of inactivated sterile human blood serum and plas mol taken in the weight ration of 1:1.

Plasmol is a pharmaceutical preparation prepared from human blood. This is a colourless or slightly yellowish transparent or slightly opalescent liquid with a specific odour.

For the purpose of differential diagnosis of gonorrhea the following investigation procedures are performed according to the present invention.

Onto discs of the polyacrylamide gel swollen in the physiological solution till their mass is increased, for example, by 4.5 times and located in Patri dishes subjected to sterilization by a conventional tech nique 0.25-0.5 ml of human blood serum is poured by means of pipette along with 0.25-0.5 ml of plas mol, whereafter inactivation is carried out also by a conventional method. Then reinoculation of a recovered day's age culture of gonococci from 20 patients suffering from acute gonorrhea obtained on a dense nutrient medium-serum agar taken as a comparison medium (of l.M.Ovchinnikov, "Laboratory Diagnosis of Veneric Diseases", Moscow, Medicina Publishing House, 1969, pp.67- 69) was effected onto the medium prepared by the above-mentioned process.The inoculated dishes are placed into a desiccator which is then put into a thermostat at the temperature of 37"C for 24 hours. Colonies of gonococci grown on the serum medium with the polyacrylamide gel were located as dew drops with a smooth surface, they were colourless and transparent. Bacterioscopy of smears prepared from these colonies and coloured according to Gram and to the "Method of Differential Diagnosis of Gonorrhea and Bacterial Urethrites" (of. USSR Inventor's Certificate No. 826208) resulted in detection of gram-negative bean-like diplococci.

In the second series of studies excretions from urethra of 30 patients with acute gonorrhea and chronical gonorrhea were inoculated onto the medium according to the present invention after bacterioscopic justification of the diagnosis (coloration according to Gram and USSR Inventor's Certificate No. 826208) and bacteriological justification (innoculation onto a medium comprising serum agar). In all cases on the medium according to the present invention good growth of gonococci was observed.

As a result of the tests, in day's age cultures on the dense nutrient medium according to the present invention a more intensive growth of gonococci was observed as compared to the growth on the prior art nutrient medium. Upon culturing on the medium according to the present invention gonococci retained their morphological, tinctorial and cultural properties. The use of this medium for culturing of gon ococci will make it possible to considerably broaden the opportunities for diagnostic methods of determination of gonorrhea, especially in chronical and torpid forms thereof.

For the purpose of growing and studying the interaction of microorganisms and cell cultures of a macroorganism in vivo and in vitro the nutrient medium according to the present invention can be prepared in the following manner.

A mixture of acrylamide and N,N'-methylene-bis-acrylamide and a radical-type initiator, for example a mixture of N,N,N',N'-tetramethylethylenediamine and ammonium persulphate prepared on a physiological solution is poured into a reactor and a polyacrylamide gel is formed during copolymerization in two flat plates, one which has recesses positioned coaxially or in a stagger-like manner.The plates are impregnated with a solution containing acrylamide and N,N'-methylene-bis-acrylamide at a weight ration thereof equal to 15.0-20.0:0.019-0.132 and aligned in such a manner that closed cavities are made for a subsequent culturing of microrganisms or cell cultures of a macroorganism therein, the plates are kept till a single block is formed which is then divided into parts so that each of them has at least one of the above-mentioned closed cavities.

These parts with closed cavities can be referred to as diffusion chambers which are kept in a physiological solution so that the polyarylamide gel (from which they are made) swells and they are increased in mass by 2.5-4.5 times. Then they are treated with a liquid nutrient medium depending on the studied microorganism or a culture of cells of a macroorganism. The equal quantitative proportions of the components in the reaction mixture employed for the formation of the plates and in the mixture for impregnation of the plates ensure uniformity of the wall structure of the resulting diffusion chambers, thus causing a uniform diffusion of substances and simplifying the process of their manufacture.

Depending on the disposition of recesses in a plate one-and multi-cavity chambers can be produced.

The distance between recesses and plate thickness can be varied depending on the chamber purpose.

Thus, in the manufacture of multi-cavity chambers recesses in the plates are positioned in a staggerlike manner, preferably at a distance of 0.5-2 mm from each other to ensure an optimal diffusion process between closed cavities of these chambers. They make it possible to simultaneously study multi-component systems during their interaction, as well as under the influence of factors of a live organism or ambient medium. To produce one-cavity chambers the plates are formed with coaxially-positioned recesses.

The chambers have been employed by the inventors for growing cells of a macroorganism and microorganisms in vivo and in vitro. To this end, into the chamber cavity cells of lymphoid organs and other organs (e.g. spleen, lymph nodes, thymus and bone marrow are introduced by means of a syringe by puncturing the chamber wall with a needle. Then the chambers are implanted into the organism of recipients. After a certain period they removed, subjected to microscopy in order to study the cell organization and character of the colonies. Cells were taken from the cavity by puncturing the chamber wall and subjected to analysis of their morphostructural and functional characteristics.For example, upon culturing of cells of bone marrow in vivo the growth and development of cells of the myeloid series was maintained for one month and longer which is substantially superior to usual terms of survival of these cells in diffusion chambers made from other materials.

Some specific examples illustrating the process according to the present invention are given hereinbelow.

Example 1 For the production of a polyacrylamide gel three solutions are prepared (A, B and C) according to the following procedure: (the starting component amounts are specified on the basis of 1,000 ml of a solution): 1. Preparation of solution A 5 ml of N,N,N',N'-tetramethylethylenediamine are dissolved in 995 ml of a 0.85% aqueous solution of NaCI. The solution is kept in a dark glassware at the temperature of 4"C.

2. Preparation of solution B 0.594 g of N,N'-methylene-bis-acrylamide is dissolved in 500 ml of a 0.85% aqueous solution of NaCI, heated to the temperature of 60"C, then added with 470 g of acrylamide which is stirred till a complete dissolution. The thus- prepared solution is filtered, brought to the volume of 1,000 ml with a 0.80% aqueous solution of NaCI. The solution is kept in a dark glass vessel at the temperature of 4"C.

3. Preparation of solution C 1.78 g of persulphate are dissolve in 100 ml of a 0.85% aqueous solution of NaCI and stored in a darl glass vessel.

A reaction mixture is prepared from the above solutions (A,B and C). To this end, solutions A, B and C are successively mixed in the following volume proportions: A:B:C = 16:31:52 The reaction mixture contains the monomers at the weight ratio of acrylamide and N,N'-methylene-bisacrylamide equal to 15.0:0.019; the weight ratio of their mixture to the physiological solution is 1:6.

The reaction mixture is poured into a 200 ml reactor and discs of a predetermined diameter are shaped from the polyacrylamide gel. The copolymerization process occurs for 40 minutes. The resulting discs are washed with a physiological solution and subjected to swelling in a 0.85% solution of NaCI till their mass is increased by 4.5 times. After swelling the polyacrylamide gel contains, per cent by mass: copolymer of acrylamide and N,N'-methylene-bis-acrylamide 3.3 physiological solution 96.7 This gel is used as a base for the production of a dense Hottinger nutrient medium. The discs are placed into a glass vessel, poured with Hottinger tryptic overcook containing 200 mg-% of amine nitrogen at the rate of 20 ml of the broth per disc and allowed to stand for 2-3 hours for saturation.The impregnated discs are sterilized by steam under pressure at the temperature of 1200C for 30 minutes. The ready-to-use nutrient media are dried and inoculated with test microorganisms St.aureus 209, E.coli M17, Sh.flexneri, Bac.cereus 504, Bac.subtilis, Candida alticans.

As the control use is made of the same cultures of microorganisms cultivated on media with the polyacrylamide gel produced according to the USSR Inventor's Certificate No. 977466. The inoculates are subjected to incubation in a thermostat at the temperature of 37"C for one day.

This nutrient medium is not damaged during preparation and use thereof; cultures of the microorganisms show a typical growth, by means of a bacteriological loop. The following Table 1 shows the result demonstrating the growth intensity of various microorganisms on the nutrient medium according to the present invention and on the medium described in the USSR Inventor's Certificate No.977466.

If follows from Table 1 that the average number of colonies grown on the dense nutrient medium according to the according to the present invention by the process disclosed herein is more than that grown on the dense nutrient medium described in the USSR Inventor's Certificate No.977466.

TABLE 1 Species and strains Inoculati- Average Average number of microorganisms on dose number of of colonies (ac (colony- grown co- cording to the forming lonies USSR Inventor's units) (as in Ex- Certificate ample 1) No.977466) Staph.aureus 209 100 98 94 E.coli K-12 100 96 92 E.coli M-17 100 94 84 Sh.flexneri 100 82 70 Bac.cereus 504 100 86 78 Bac.subtilis 100 88 74 Candida albicans 100 78 70 Example 2 Solutions A and C are prepared in a manner similar to that described in Example 1 hereinabove. For the preparation of solution B 0.844 g of N,N'-methylene-bis-acrylamide is dissolved in 500 ml of a 0.85% aqueous solution of NaCI, heated to the temperature of 60"C and added with 470 g of acrylamide. The solution is filtered and brought to 1000 ml with a 0.85% solution of NaCI.

The reaction mixture is prepared as in Example 1.

The reaction mixture contains acrylamide and N,N'-met-hylene- bis-acrylamide in the weight ratio of 15.0:0.027; the weight ratio of their mixture to the physiological solution is equal to 1:7. The copolymerization process is conducted in a manner similar to that described in Example 1. The resulting discs of the polyacrylamide gel are subjected to swelling in Hanks physiological solution till its mass is increased by 3.2 times. After swelling the polyacrylamide gel contains, per cent by mass: copolymer of acrylamide and N,N'-methylene-bis-acrylamide 4.0 physiological solution 96.0 In the present Example for impregnation of the gel a meat-peptone broth is used. The impregnation and sterilization are carried out as described in Example 1. The test-microorganism is a culture of Staureus, strain 209.As the control, the same culture grown on the dense nutrient medium described in the USSR Inventor's Certificate No.977466 and on the meat-peptone agar is used. After a 24-hours' incubation of the inoculates in a a thermostat at the temperature of 37"C a more intensive growth of the staphylococcus culture is observed on the nutrient medium according to the present invention than on the control inoculations.

The colonies of staphylococcus are typical -round, smooth, glossy, oily with an intensive pigmentation.

The typicity of the culture is proven by microscopic analysis. The colonies are fully removed by means of a bacteriological loop without damaging the surface of the dense nutrient medium.

Example 3 Solutions A and C are prepared as described in Example 1. The solution B is prepared in the following manner.

Into a metering 1,000 ml flask 0.937 g of N,N'-methylene- bis-acrylamide is introduced, added with 400 ml of a 0.85% solution of NaCI. After a complete dissolution of the monomer the solution is heated to the temperature of 60C and then 625 g of acrylamide are added. The solution is filtered and brought to 100 ml with a 0.85% solution of NaCI.

The reaction mixture contains acrylamide and N,N'-methylene- bis-acrylamide at the weight ratio of 20.0:0.03 respectively, while the weight ratio of their mixture to the physiological solution is equal to 1:4.

The process of copolymerization is conducted in a manner similar to that described in Example 1.

The resulting discs of the polyacrylamide gel are subjected to swelling in a physiological solution (Earl solution) till they are increased in mass by 3.7 times. After swelling of the polyacrylamide gel it contains, per cent by mass: copolymer of acrylamide and N,N'-methylene-bis-acrylamide 5.3 physiological solution 94.7 As the liquid nutrient medium for impregnation use is made of wort; the impregnation and sterilization are carried out as described in Example 1 hereinbefore. After sterilization the dense nutrient medium does not change its properties. The test microorganisms is a culture of the fungus Fusarium avenaceum.

After 4 days of culturing in a thermostat at the temperature of 28"C typical fungal colonies appeared with a characteristic fluffy mycelium having pink colour. Microscopic studies show that the mycelium has a characteristic structure and sporing is observed. The colonies are readily removed by means of a bacterilogical loop without damaging of the nutrient medium surface.

Example 4 Solutions A and C are prepared as described in Example 1 hereinbefore.

Solution B is prepared in the following manner.

4.125 g of N,N'-methylene-bis-acrylamide are dissolved in 500 ml of a 0.85% aqueous solution of NaCI, heated to the temperature of 60"C, then added with 470 g of acrylamide and stirred to a complete dissolution. The resulting solution is filtered, brought to 1,000 ml with a 0.85% aqueous solution of NaCI.

From the prepared solutions the reaction mixture is obtained and copolymerization is carried out under conditions similar to those described in Example 1 hereinbefore.

The reaction mixture contains acrylamide and N,N'-methylene- bis-acrylamide at the weight ratio thereof of 15.0:0.132 while the weight ratio of their mixture to the physiological solution is equal to 1:5.

This polyacrylamide gel is used as base for a dense nutrient medium for the production of a biomass of Staphylococcus aureus 209 P. The produced discs are washed with a 0.85% solution of NaCI, immersed into Hottinger tryptic overcook containing 200 mg-% of amine nitrogen till the gel mass is increased by 2.5 times.

The final dense nutrient media are sterlized by steam under pressure at the temperature of 1200C for minutes. As the control an agarized Hottinger medium is used which contains 200 mg-% of amine nitrogen and the medium described in the USSR Inventor's Certificate No. 977466. Staphylococcus is inoculated onto a disc with a diameter of 60-63 mm at the rate of 108 microbal bodies in 1 ml. Table 2 shows the data illustrating the yield of the biomass of staphylococcus aureus on different dense nutrient media.

TABLE 2 Biomass of Staphylococcus, mgicm2, obtained on dense nutrient media of this Example Example 1 USSR Inventor's on agarized Certificate medium No.977466 2.01 1.57 1.50 1.53 After swelling the polyacrylamide gel contains, per cent by mass: copolymer of acrylamide and N,N'-methylene-bis-acrylamide 6.0 Hottinger tryptic overcook 94.0 Example 5 The polyacrylamide gel is produced as described in Example 1 hereinbefore.

This polyacrylamide gel is used as a base for a dense nutrient medium for culturing fungus Verticlllium dahliae. The discs washed in a 0.85% solution of NaCI are placed into a liquid nutrient medium-wort and subjected to swelling till their mass is increased by 3.5 times.

After sterilization the final dense nutrient media are inoculated with a culture of the fungus. As the control a culture of fungus Verticillium dahliae grown on an agarized nutrient medium impregnated with wort and on the medium prepared as in Example 1 is used. The inoculates are subjected to incubation at the temperature of 28"C.

On the medium prepared according to Example 5 an intensive growth of the fungus is observed which has characteristic morphological and cultural properties on the 3-rd day, whereas on the control media the growth of a similar intensity is observed only on the 5-th day.

Example 6 A reaction mixture prepared as described in Example 1 is cast into a reactor and a polyacrylamide gel is shaped in two flat plates. One of them has the dimensions of 180x100x0.8 mm and the second with the dimensions of 180x100x2.5 mm has recesses of 3x1.0 mm size positioned in a stagger-like manner at a distance of 1-1.5 mm from one another. The sterilization process is carried for 40 minutes. Then the thus produced plates are impregnated with a mixture having composition similar to the reaction mixture described in Example 1. Then the plates are aligned so as to form close cavities for a subsequent cultivation, therein, of cultures of cells of macro-and microorganisms. The plates are kept for 40 minutes till a single block is formed.Then it is divided into parts each containing three closed cavities, i.e. diffusion chambers which are placed into a 0.85% solution of NaCI and maintained therein till their mass is increased by 4.5 times. Thereafter the chambers are subjected to sterilization for 1-1.5 hours and saturated with the medium 199.

Into one of the cavities of a three-cavity diffusion chamber cells of bone marrow are placed in medium 199 in the dose of 2x106; into another -a suspension of staphyloocccus strain Cowan in a physiological solution in the dose of 2x105. Then the chambers are implanted into the peritioneal cavity of syngeneous animals.

During the first 3 days in the zone of growth of myeloid series cells colonies of macrophages and granulocytes prevail. Macrophages have different forms and size.

By the 7-th day the number of colonies of macrophages is sharply increased, cytoplasmatic bridges started to appear between macrophages and lymphocytes, cells of connecting- tissue type, histiocutes and cells associated with osteoblasts. Colonies of reticular cells and neutrophiles are formed.

In the second cavity typical round, smooth colonies of staphylococci appeared and their number in creased with prolongation of the observation period. They merged and formed a solid growth zone in the cavity.

Example 7 Solutions A and C are prepared as described in Example 1, solution B -as in Example 2.

The reaction mixture contains acrylamide and N,N'-methylene-bis-acrylamide at the weight ratio of 15.0:0.027, while the weight ratio of their mixture to the physiological solution is equal to 1:7. The reaction mixture is poured into the reactor and a polyacrylamide gel is formed as two flat plates: one with the dimensions of 120x75x2.2 mm with recesses of 4x1.2 mm positioned coaxially at the distance of 1.7 mm from one another.

The process of copolymerization is carried out for 40-50 minutes. The resulting plates are impregnated with a mixture having composition similar to that of the reaction mixture. Then the plates are adjusted so as to form closed cavities. They are kept for 60 minutes till a single block is formed. It is then divided into parts, each containing one closed cavity, i.e. diffusion chambers are produced which are then washed with a physiological solution, kept in a physiological solution of NaCI till their mass is increased by 2.8 times.

Thereafter the chambers are subjected to sterilization for 1-1.5 hours and saturated with medium 199.

Bone marrow is extracted from femur of intact syngeneous animals. The cells of bone marrow in medium 199 in the volume of 0.2 ml (4x106 cells) are placed into diffusion chambers which are implanted into the peritoneal cavity of syngeneous recipients.

After culturing of cells in vivo the diffusion chambers are removed from peritoneal cavity and tested under microscope to analyze the monolayer of cells (growth zone, cell composition), as well as smearsprints produced from grown cell cultures.

On the 3-rd day of culturing of bone marrow cells in the diffusion chambers implanted into the peritoneal cavity of normal animals a heterogeneous cell composition was noted which was characteristic of intact bone marrow. Later on, (on the 7-day) certain portion of cells degenerated, while the other started to transform into a certain form. Various stages of differentiation of granulocytes were observed. Metamyelocytic cells were arranged in groups near served. Metamyelocytes cells were arranged in groups near well-developed fibres of a non-differentiated tissue and had protrusions on their surface. Mature granulocytes had a nucleus characteristic for neutrophiles. The intermediate forms have a clearly pronounced nuclear segmentation. In the growth zone large-size epithelioid cells (40 pwm) were formed with insignificant bulbs on their surface.They were located in groups in the monolayer as well as individually.

Their nucleus had a flattened appearance and phagocytized inclusions were absent in cytoplasm.

Monocytes turned to be very viable. Large phagocytic vacuoles were frequently filled with contents.

Monocytes were well classified by their bean-like form of the nucleus frequently positioned excentrically.

Upon a longer culturing the nucleus had a more round shape. Monocytes located frequently near epithelioid cells, though without any clearly pronounce liability to aggregation.

In 21-days' age culture the growing cells were located in the cavity of the diffusion chamber, thus creating a specific structural organization. The superficial layer consisted of epithelioid cells, followed by a group of monocytic, fat cells and young forms of neutrophiles. Mature forms were arranged along the periphery. Lymphocytic and blastic cells were located inside the adhesive layer. In the growth zone fat cells with a small dense nucleus were observed. In some cases two nuclei forms occurred. They were well identified on electronograms by fine fat drops in the cytoplasm.Lipid inclusions were frequently located in nest-like arrangements, sometimes merging into drops with a diameter of up to 40 m. As a result of accumulation of fat these cells sometimes had size of up to 120 ssLm. A unit vacuolar membrane pointed to the intracellular synthesis of lipid. A round or oval nucleus consisted of condensed chromatin.

Cytoplasm of mature cells was with numerous mitochrondrions. At the stage of early accumulation of fat the cytoplasm was indifferentiated. Fat cells had a trend towards arrangement in groups, forming large foci between granulocytic colonies and for a long time retained a functional relationship.

Cells of megakaryocytic series were also observed. These were large-size spherical cells sometimes with protrusions on the surface of the cytoplasmatic membrane. No active thrombocytopoesis was noticed.

Example 8 Solutions A and C are prepared as described in Example 1 hereinbefore, while solution B is prepared as in Example 4.

The reaction mixture contains acrylamide and N,N'-methylene- bis-acrylamide at the weight ratio thereof equal to 15.0:0.132, while the weight ratio of their mixture to the physiological solution is 1:5.

The reaction mixture is poured into a reactor and polyacrylamide gel is formed into two flat plates. One plate has the size of 150x80x1.2 mm, while the other with the size of 150x80x2.5 mm has recesses with the size of 5x1.3 mm positioned coaxially at the distance of 1.8 cm. The process of copolymerization is conducted for 40 minutes. The resulting plates are impregnated with a mixture having composition similar to that of the reaction mixture and adjusted so as to form closed cavities.

They are kept for 50 minutes till a single block containing closed cavities is formed. Then the block is divided into parts containing each one closed cavity, i.e. diffusion chambers, which are placed into a physiological solution of NaCI till their mass is increased by 3.2 times. Thereafter the chambers are sterilized and impregnated with Hanks solution.

For cultivation of leucocytes of a healthy person, into the cavity of a diffusion chamber a population of leucocytes containing all forms of cells of white blood in Hanks solution is introduced into the diffusion chamber cavity. Then the diffusion chambers were implanted into the organism of xenogeneous animals and removed within the required observation period. The experiments have shown that lymphocytes were preserved for 20 days. However, their number reduced from 79 to 20%. Monocytes and neutrophiles were viable only during the first 2-3 days. An increased amount of macrophages was noted f-om 15% on the 2-nd day to 50% on the 20-th day. Blasts, fibroblastoid and transition cells started to appear in the culture from the 8-th day.

Claims (8)

1. A process for producing a dense nutrient medium for culturing microorganisms and cell cultures of a macroorganism comprising copolymerization of acrylamide and N,N'-methylene- bis-acrylamide at a weight ratio equal to 15.0-20.0: :0.019-0.132 respectively in a physiological solution in the presence of a radical-type initiator till the formation of a polyacrylamide gel, followed by washing thereof with a physiological solution and keeping in the physiological solution till its mass is increased by 2.8-4.5 times with a subsequent treatment by a liquid nutrient medium, or subjecting the polyacrylamide gel resulting from washing to swelling in a liquid nutrient medium till its mass is increased by 2.5-3.5 times.

2. A process according to Claim 1, wherein said copolymerization is conducted at a weight ratio of the mixture of acrylamide and N,N'-methylene-bis-acrylamide to the physiological solution equal to 1:4-7 respectively.

3. A process according to Claims 1 and 2, wherein as the physiological solution a 0.85% aqueous solution of sodium chloride is used, or a Ringer-Lock solution, or Earl solution, or Hanks solution, or a 5% solution of glucose.

4. A process according to Claim 1, wherein as the liquid nutrient medium Hottinger tryptic overcook, Marten broth, meat-peptone broth, peptone water, casein hydrolyzate wort, or medium 199 is used.

5. A process according to Claim 1, wherein for culturing gonococci, the polyacrylamide gel swollen in the physiological solution is treated with a liquid nutrient medium comprising a mixture of inactivated human blood serum and plasmol taken in the weight ratio of 1:1.

6. A process according to Claim 1, wherein the polyacrylamide gel resulting from copolymerization is shaped into two flat plates, one having recesses positioned coaxially or in a stagger-like manner, whereafter the plates are impregnated with a solution containing acrylamide and N,N'-methylene- bis-acrylamide at a weight ratio of 15.0-20.0:0.19-0.132 respectively and adjusted so as to form closed cavities for a subsequent cultivation of microorganisms or cell cultures of a macroorganisms therein, kept till the formation of a single block, followed by division thereof into parts having each at least one of said closed cavities.

7. A process for producing a dense nutrient medium for culturing microorganisms and cell cultures of a macroorganism, substantially as described herein with reference to any one of Example 1 to 8.

8. A dense nutrient medium for culturing microorganisms and cell structures of a macroorganism, as produced by a process according to any preceding claim.