HU193585B - Process for production of microorganisms and medium suitable for growing of cell-culture of microorganisms - Google Patents

Abstract

The present invention relates to a solid culture medium for culturing microorganisms and microorganisms of cell cultures by copolymerization of acrylamide and N, N'-methylene-bis-acrylamide in physiological saline, in the presence of a radical initiator, and washing of the resulting polyacrylamide gel with a physiological solution such that: (15.0 20.0): copolymerize acrylic amide Ν, Ν-methylene-bis-acrylic amide monomer mixture (0.019 to 0.132), wash with a physiological solution and wash out the polyacrylamide gel after washing (a) physiological with a liquid medium, or b) swollen with a liquid medium until its weight increases 2.5 to 3.5 times. -1-

Description

The present invention relates to a process for the production of solid media for culturing cell cultures of microorganisms and macroorganisms.

Currently, in microbiological practice, agar-agar is generally a natural gel-based solid medium which is very expensive and often deficient in use. Therefore, developments in the development of synthetic-based gel varieties replacing agar-agar in the microbiological field are very topical.

From U.S. Patent No. 659,619, there is known a process for culturing microorganisms in solid medium by copolymerization of prylamide and N, N'-methylenebisacrylamide followed by aqueous washing of 1 (Spotolyl lactide) gel with soaked in nutrients and sterilized.

The main advantage of using the polyacrylamide gel as a substrate base is that it has a stable composition, suitable after washing with water, and does not contain substances that would adversely affect the cultivation of microorganisms and does not require clarification before use. The polyacrylamide gel produced by the process of the above-mentioned patent and used as a solid medium is quite hard, due to the high cross-linking of the polyacrylamide and makes it difficult to diffuse the high molecular weight fractions of the nutrient into the carrier. The removal of cultured microorganisms from such media can only be accomplished by rinsing with saline.

US-A-977466 discloses another process for the production of a polyacrylamide gel, also for biological and medical purposes, which can be used as a base for solid media. According to this procedure, the copolymerization of acrylamide and Ν, Ν'-methylene-bis-acrylamide is carried out in a 0.5% sodium chloride solution in the presence of a radical initiator, and the starting materials are 0.5 to 40.0: 2.5 to 12 , 0 in weight ratio.

The resulting polyacrylamide gel, which contains the maximum amount of water after completion of the copolymerization, is washed with 0.5% sodium chloride solution for 12 hours to remove unreacted monomers, and the solution is changed every 4 hours. The resulting polyacrylamide gel has the following composition:

polyacrylamide 3.0-28% by weight

0.5% sodium chloride solution, 72.0 to 97.0% by weight

The resulting polyacrylamide gel is soaked in nutrient media suitable for culturing various microorganisms, such as meat-peptone broth substrates.

The solid medium prepared according to the above procedure is free of toxic monomers 2 and can be used to study the biological properties of various microorganisms, animal and human cells.

However, the disadvantages of gels prepared according to the above process are that their hardness makes their surface very vulnerable to various operations, such as sterilization, inoculation with microorganisms or bacteriological stripping of colonies. Furthermore, the microorganisms can be removed after repeated inoculation only by rinsing with saline, which is not advantageous in most microbiological workflows. This process is particularly complicated in the case of long cultivation of microorganisms.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a process for producing solid media suitable for culturing microorganisms and cell cultures which, by virtue of their physico-chemical properties, can broaden the field of microbiological application.

The object of the present invention can be accomplished by a process wherein acrylamide is prepared with Ν, Ν'-methylene-bis-acrylamide in physiological saline in the presence of a radical initiator, starting from 15.0 to 20.0:

: 0.019-0.132% (w / w) copolymerize and after washing wash the excellent polyacrylamide gel in saline until its weight is 2.8-4.5 times. the polyacrylamide gel which precipitates after washing is swollen in a liquid medium 2.5 to 3.5 times its weight.

The solid medium produced by the process of the present invention is resistant to various influences, is not damaged during various operations, has a density close to that of agar, has good elastic and adhesive properties (adheres well to a Petri dish). Compared to known solid media, the solid media produced by the process of the present invention exhibits good slip properties when inoculated and microorganisms are removed and its surface is not vulnerable.

Microorganism cultures are well dispersed on the surface of a new type of solid medium without ingestion, thus providing favorable living conditions, which can increase biomass yield. In order to increase the quality of the solid medium during copolymerization, the weight ratio of the starting mixture of acrylamide and N, N'-methylene-bis-acrylamide to the physiological solution during the polymerization reaction creates a precondition for the formation of a three-dimensional, cross-linked polyacrylamide gel. it provides the physical-mechanical properties of the solid medium to be formed.

In order to broaden the microbiological application of the solid media produced by the process of the invention, it is advantageous to use 0.85% saline as a saline solution.

-2193585 aqueous sodium chloride solution, Ringer-Locke, Earl, Hanks solution (for composition see

D. Pol: "Cell and Tissue Cultures, Medgiz Publishers, 1963, p. 96), respectively. 5% aqueous glucose solution and Hottinger's trypsin digestion solution as a liquid medium, Marten's broth medium (for composition, see JA Kozlov, "Mediums for Medical Microbiology, Medgiz, 1950, pp. 51-52 and 63), peptone broth medium, peptone water solution, casein hydrolyzate, saline or 199 medium (for composition see D. Pol, "Cell and Tissue Cultures", Medgiz, 1963, p. 103).

In the case of culturing gonococci, the swollen polyacrylamide gel in saline is suitably treated in a liquid medium which is a 1: 1 mixture of inactivated, sterilized human blood serum and human blood derived plasma product. The solid medium thus produced provides high quality cultures of gonococcal cultures while maintaining morphological, dye culture and other biological properties. The use of this solid medium increases the chances of detection and bacteriological diagnosis of gonorrhea, particularly in chronic and torpid (reny) forms. The production time of this solid medium is significantly greater than that of other solid media suitable for culturing gonococci (e.g., ascites meat peptone agar, Baily agar (for composition see JA Kozlov, cited above, p. 63), serum agar). shorter and many costly components can be eliminated.

In order to expand the field of application of the solid media according to the invention, the polyacrylamide gel may be formed in two flat strips during copolymerization. One of these is cavities formed in a coaxial or chessboard-like arrangement, and these strips are soaked in solutions of acrylamide and Ν, Ν'-methylene-bis-acrylamide in a ratio of 15.0 to 20.0: 0.019-0.132, and so on. bringing them together to form closed cavities for the cultivation of microorganisms and cell cultures. The interlaced strips are thus held until a uniform block is formed and then separated into sections each containing at least one cavity. The resulting pieces were swelled in saline and treated with liquid medium.

The cavities formed in this way function as diffusion chambers and, due to the advantageous properties of the polyacrylamide gel, exhibit good strength, elasticity and heat resistance. Easy to reproduce, long-term storage, sterilization. This solid medium in the form of a diffusion chamber is widely applicable for both in vivo and in vitro use and is atraumatic when transplanted into an organism such as the abdominal cavity of an animal. They are inert to the organism, which is very important when examining the interaction between objects introduced into the ventricular cavity and various environmental factors.

Due to the advantageous properties of the polyacrylamide gel, the diffusion chambers produced therefrom exhibit very good diffusion properties, which allow the necessary nutrients and other substances to reach the desired cell culture in the chamber cavity and exert their desired effect. A further advantage of these objects is that they are transparent, such as the cell cultures to be examined and the cells. microorganisms can be observed microscopically.

Solid media suitable for culturing microorganisms are prepared as follows. First, a reaction mixture is prepared which contains the starting monomers in physiological saline and has a weight ratio of acrylamide to Ν, Ν'-methylene-bis-acrylamide of 15.0 to 20.0: 0.019 to 0.132.

As a saline solution, the monomer mixture is 4: 1 to 7, respectively. 0.85% or 0.9% sodium chloride solution, 5% glucose solution in water, Ringer-Locke, Hanks or Earl solution are used. By varying the weight ratio of the starting monomer mixture and saline, different types of polyacrylamide gels are obtained. All of them are more elastic than the known gels and provide a simpler and high quality medium.

Copolymerization of the starting monomers may be carried out with or without heating, using known initiators. The reactor material used may be glass, metal, ceramic or plastic. During the copolymerization reaction, the resulting polyacrylamide gel takes the form of a reactor. After copolymerization, the resulting polyacrylamide gel is washed with saline, swelled in saline 2.8 to 4.5 times its weight and treated with the liquid medium. Trypsin digestion Hottinger's solution, Marten's broth, meat-peptone-broth solution, peptone-water solution, casein hydrolyzate, saline and 199 medium can be used for this purpose.

In another embodiment of the process of the invention, the polyacrylamide gel washed with saline is directly swollen in a fluid medium of 2.5 to 3.5 times its weight and then the swollen polyacrylamide gel is sterilized. The solid medium thus obtained is ready for use and has the advantage that the biomass yield is very good.

The composition of the liquid medium used is the microorganism conc3

-3193585 depends on the nutritional requirements of the group of meadows. Any known liquid medium that meets the nutritional requirements of known microorganisms and cells, including natural, semi-synthetic and synthetic substrate compositions and mixtures thereof, can be used to swell the polyacrylamide gels of the present invention. The solid media produced by the process of the invention for cultivation of microorganisms and cell cultures have good adhesion properties (good adhesion to Petri dishes), various resistance to action, no damage during operations, good slip of bacteriological duck during inoculation and removal of microorganisms. , without damaging its surface.

Microorganism cultures are well distributed on the surface of the medium without being ingested, creating optimal conditions for the microorganisms to function, thereby increasing biomass production.

The above-mentioned properties of the solid media are provided by the expanded polyacrylamide gel and, as mentioned above, can be advantageously used for culturing gonococcal cultures. Preferably, the polyacrylamide gel, swelled at 4.5 times its weight in saline, is treated with a liquid medium which is a mixture of inactivated, sterile human blood serum and plasmol.

The plazmol, colorless or slightly yellowish transparent or weakly characteristic odor pharmaceutical composition in the human blood, which has been sterilized at 100 ° C and 1.2 to 10 ⁵ psi.

The differential diagnosis of gonorrhea is as follows.

Polyacrylamide disks were swelled 4.5 times in saline, placed in a Petri dish, sterilized in a known manner, pipetted with 0.25-0.5 ml of human blood serum, 0.25-0.5 ml of plasma and inactivated in known manner. Subsequently, inoculated cultures of selected gonococcal cultures from 20 patients with acute gonorrhea are again inoculated using this agar serum medium (I. M. Ovesinikov: Laboratory Diagnostics, Moscow, Medicina Publisher, 1967, 67-69). Petri dishes containing the inoculum were then placed in a desiccator and thermostated for 24 hours at 37 ° C. Gonorrhea colonies cultured on polyacrylamide gel are distributed on the serum in a form similar to a smooth surface dew drop, colorless and translucent. Gram-negative, bean-like diplococcus can be detected in bacterial scintillation by sulfur prepared from colonies. (Colonization was performed according to Gram and 826208, "Procedure for the differential diagnosis of gonorrhea and bacterial ureteritis.")

In another assay, the inoculation medium according to the invention was administered 30 or more times. was performed on ureter samples from a patient with chronic gonorrhea. Diagnosis was confirmed by bacterioscopy (Gram staining or US Patent No. 826208) and bacteriological procedure (inoculation into serum agar). Gonorrhea strains were again bred with good results in the culture medium according to the invention.

As a result of our experiments, it has been found that the growth of daily cultures on the medium according to the invention is much more intensive than on other known media. Gonococcal colonies retained their morphological, dye, and culture properties as they grew on the medium of the invention. By using these media, diagnostic methods for gonorrhea, especially its torpid and chronic forms, can be significantly expanded.

For the study of the interaction of microorganisms and macroorganism cell cultures and their in vivo and in vitro mechanism of action, the culture medium for cultivation is prepared as follows.

A mixture of acrylamide and N, N'-methylene bis-acrylamide and a radical initiator such as a mixture of tetramethylenediamine and ammonium peroxodisulfate is copolymerized in a reactor in a reactor. The resulting polyacrylamide gel is formed into two flat strips, one of which forms cavities in a coaxial or chessboard arrangement. These strips are then impregnated with a mixture of acrylamide and N, N'-methylene-bis-acrylamide solutions in a weight ratio of 15.0 to 20.0: 0.019 to 0.132, and superimposed to form closed cavities, which are then microorganisms or microorganisms. cell cultures. The strips are then allowed to stand until a single block is formed and then separated into sections each containing at least one cavity.

These small cavities are considered as diffusion chambers, which are swollen 2.5 to 4.5 times their volume in physiological saline, and are thus the microorganism to be tested. liquid culture medium selected according to cell culture. The fact that an identical mixture reaction mixture was used to form the strips and subsequently convert them ensures that the diffusion, chamber structure, and wall thickness are uniform, allowing uniform diffusion of the materials.

Depending on the arrangement of the cavities, strips having only one or several cavities may be formed. The distance between the cavities and the thickness of the strip depends on the application. Preferably, for strips comprising multiple cavities, the cavities have a spacing of 0.5 to 2.0 mm and are arranged in a chessboard-like manner. This is optimal,

-4193585 allows diffusion processes between separated chambers. At the same time, it is possible to study the interaction between each other or the environment and the various effects on living organisms in multi-component systems. For chambers containing a cavity, the cavities are coaxially formed.

The chambers prepared as described above are used for in vivo and in vitro studies of cells of macro and microorganisms. To this end, for example, cells in lymphoid organs (e.g., spleen, lymph node, thymus, bone marrow) are inserted into the chamber cavity using a needle, and the chamber is implanted into the recipient organ. After a fixed period of time, cellular organization and culture characteristics are harvested and observed microscopically. By puncturing the ventricular wall, cells can be removed from each well and examined for morphology and functional properties. In vivo growth of bone marrow cells was followed by cell growth for 1 month and was found to be significantly longer than the survival time of chambers made of other material.

The following examples illustrate the process of the invention in more detail.

Example 1

For the preparation of the polyacrylamide gel, three solutions (A, B, C) are prepared as follows (in each case, the indicated amounts of starting materials per 1000 ml of solution):

Preparation of solution A ml of Ν, Ν, Ν ', Ν'-tetramethylethylenediamine is dissolved in 995 ml of 0.85% aqueous sodium chloride solution and stored at 4 ° C protected from light.

B) Preparation of solution

Dissolve 0.594 g of N, N'-methylene bisacrylamide in 500 ml of 0.85% aqueous sodium chloride solution, add 470 g of acrylamide and heat at 60 ° C until completely dissolved. At this time, the solution was filtered and made up to 1000 ml with 0.85% aqueous sodium chloride. The solution was protected from light and stored ^at 4 ° C.

C) preparation of a solution

1.78 g of peroxysulphate are dissolved in 100 ml of 0.85% aqueous sodium chloride solution and stored protected from light.

From the solutions prepared as above, prepare the reaction mixture by mixing them in the following ratio:

A: B: C = 16: 31: 52.

In such a mixture of solutions, the weight ratio of the monomers to acrylamide: N, N'-methylene-bis-acrylamide = 15.0: 0.019 and the weight ratio of the monomer mixture to physiological saline is 1: 6.

The above reaction mixture was poured into a 200 ml reactor and polyacrylamide gel discs of a certain diameter were prepared. The polymerization reaction was completed in 40 minutes. The resulting discs were washed with saline and swollen to 4.5 times their weight in 0.85% sodium chloride solution. The polyacrylamide gel then has the following composition:

Acrylamide-N, N'-methylene-bisacrylamide copolymer 3.3% by weight

Saline was 96.7% by weight.

The above-described gel is then made into a Hottinger solid medium. To do this, the discs were placed in a glass jar, and the Hottinger's trypsin digestive broth containing 200 mg% aminonitrogen was poured into each of the disks with 200 ml of solution and allowed to stand for 2-3 hours. The impregnated discs are then sterilized by steam at 120 ° C for 30 minutes under pressure. That's it. the ready-to-use medium is inoculated with the microorganisms to be tested. The following strains were used in the experiment: St. aureus 209, E. coli K-12,

E. coli Μ-17, Sh. flexneri, Bac. cereus 504, Bac. subtilis, Candida albicans.

As a control, the above microorganisms are also cultured on a medium prepared from a polyacrylamide gel prepared according to the procedure described in US Patent No. 977466.

The media were not damaged during use, the microorganisms showed a characteristic growth, did not grow into the medium and were easily removed by bacteriological piglets. The following Table 1 summarizes the growth rates for the various microorganisms as determined by the method according to the invention as well as the media prepared according to the method of the US Patent No. 977466.

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Table 1

Type of strain of microorganism Vaccination volume (colony forming unit) The formed colonies average number (Example 1 of nutritional oils) Mean number of colonies formed No. 977466 times. certified nutrient oil Staph. aureus 209 100 98 94 E. Coli K-12 100 96 92 E. Coli V-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

As can be seen from the data in this table, the growth media of the solid medium produced by the process of the present invention have a higher growth rate than the control medium according to US Patent 977466.

Example 2 30

Solutions A and C were prepared as described in Example I. To obtain solution B, 0.844 g of N, N'-methylene bisacrylamide is dissolved in 500 ml of 0.85% aqueous sodium chloride solution, heated to 60 ° C and dissolved in 35 470 g of acrylamide, filtered and Make up to 1000 ml with 85% sodium chloride solution.

The reaction mixture was prepared as described in Example 1. 40

The above reaction mixture thus contains the acrylamide and Ν, Ν'-methylene-bis-acrylamide monomers in a weight ratio of 15.0: 0.027 and the weight ratio of the monomer mixture to physiological solution is 1: 7. The copolymerization proceeds as described in Example I. place. The resulting polyacrylamide gel discs were swelled in Hanks saline solution 3.2 times their weight, whereby the polyacrylamide gel had the following composition:

Acrylamide-N, N'-methylene-bis-acrylamide copolymer 4.0% by weight

Saline 96.0% by weight

The discs made from the above gel were then soaked in meat-peptone broth medium as described in Example 55.1. Staphylococcus aureus strain 209P was used as test microorganism. As a control, cultures of the aforementioned microorganism were cultured on 60 solid media and meat peptone agar medium prepared according to US Patent 977466. After inoculation and then incubation in the thermostat for 24 hours at 37 ° C, the Staphylococcus6

culture was observed as in the control medium.

Staphylococcus colonies have a characteristic, round, smooth, shiny, oily, intense pigmentation. Observations were made microscopically. Removal of colonies with bacteriological duck does not damage the surface of the medium.

Example 3

Solutions A and C were prepared as described in Example 1. To prepare solution B, place 0.937 g of Ν, Ν'-methylene bisacrylamide in a 1000 ml volumetric flask and dissolve in 400 ml of 0.85% saline, heat to 60 ° C and add 625 g of acrylamide after complete dissolution. make up to 1000 ml.

The reaction mixture contains the acrylamide and N, N'-methylene-bis-acrylamide monomers in a weight ratio of 20.0: 0.03 and a weight ratio of the mixture to physiological saline of 1: 4. The copolymerization reaction proceeds as described in Example 1.

The resulting polyacrylamide gel discs were swollen to 3.7 times their weight in Earl saline. The polyacrylamide gel then has the following composition:

Acrylamide-N, N'-methylene-bis-acrylamide copolymer 5.3% by weight

Saline was 94.7% by weight.

Salt mixed liquid medium was used to impregnate the polyacrylamide gel discs, and the procedure and sterilization were performed as described in Example 1. The properties of the solid medium remain unchanged after sterilization. Fusarium avenaceum fungal cultures were used as test microorganisms in this experiment. On the 4th day of breeding, characteristic pink colonies appear with dense colonies of fungi. Microscopic examination shows the characteristic structure and spore formation of these colonies. The colonies can be easily removed by bacteriological duck-6193585 and the surface of the medium is not damaged,

Example 4

Solutions A and C were prepared as in Example I. To prepare solution B, 4.125 g of N, N'-methylene bisacrylamide are dissolved in 500 ml of 0.85% aqueous sodium chloride solution, heated to 60 ° C, and 470 g of acrylamide are added and stirred until completely dissolved. Filter and make up to 1000 ml with 0.85% aqueous sodium chloride solution.

Preparation of the reaction mixture and copolymerization from the above solutions was carried out as described in Example 1.

The reaction mixture thus contains the acrylamide and N.N'-methylene-bis-acrylamide monomers in a weight ratio of 15.0: 0.132 and a weight ratio of the mixture to physiological saline of 20: 1: 5.

The above polyacrylamide gel is used as a base for solid media for Staphylococcus 209P biomass. The resulting polyacrylamide discs were washed in 0.85% sodium chloride solution and swelled with Hottinger's trypsin digestion broth containing 200 mg% aminonitrogen 2.5 times their weight.

The final solid medium is sterilized by steam at 120 ° C for 30 minutes under pressure. The control medium is agarized Hottinger's medium (containing 200 mg% of aminonitrogen) and solid medium prepared according to the procedure described in US Patent No. 977466. 1-1 60-63 mm from pulley diameter of 10 ^-8 microbes / ml was inoculated with 1 Staphylococcus. The following Table 2 summarizes the results obtained, i.e., the amounts of Staphylococcus biomass obtained on different media.

Table 2

Staphylococcus biomass 2 in mg / cm in According to this example Example 1 977466 Agarizált produced nutrition produced nutrition Soviet medium on ground ground culture medium 2.01 1.57 1.50 1.55

The composition of the exemplary polyacrylamide gel after swelling is as follows:

Acrylate-Ν, Ν '-methylene-bis-acrylamide copolymer 6.0 wt.

Hottinger's trypsin digestion broth solution was 94.0 wt

Example 5

The polyacrylamide gel was prepared as described in Example 1 and used as a solid medium for culturing Verticilium dahliae in fungi. The polyacrylamide gel discs were washed with 0.85% sodium chloride solution and then swelled to 3.5 times their weight in saline medium. 55

After sterilization, the medium is inoculated with the fungal culture. As a control, agarized medium saturated with saline was prepared as in Example 1, θθ Incubation was performed at 28 ° C.

Media prepared according to Example, the solid exhibited a very intense fungal growth third day of culture in addition to the characteristic morphological and cultural properties, while similar to the growth media of a contrast roll ⁶⁵ was observed on the fifth day of culture.

example

The reactions prepared in Example 1 were poured into a reactor and formed into two flat strips of the resulting polyacrylamide gel. One of these is 180 x 100 x 0.8 mm and the other is 180 x 100 x 2.5 mm, the latter forming 3 x 1.0 mm cavities in a chessboard-like arrangement of 1 to 1.5 cm apart. mm. Sterilization is carried out for 40 minutes and the two strips are soaked in the reaction mixture corresponding to the composition described in Example 1 and placed in such a manner that closed cavities are formed for the cultivation of macro and microorganisms. Then, after 40 minutes, a single block is formed which we choose to split,

-7193585 to have three cavities each. The diffusion chambers thus formed are then swelled to 4.5 times their weight in 0.85% sodium chloride solution, then sterilized for 1 to 1.5 hours and soaked in medium 199.

Into one of the three cavities of the diffusion chamber was placed 2 x 10 ⁶ bone marrow cells distributed in medium 199, the other into 2 x 10 ⁵ staphylococcus Cowan strains suspended in physiological saline, and the diffusion chambers were implanted into the syngeneic animal.

For the first three days, macrophage and granulocyte colonies were observed in the growth zones of myeloid cells. Macrophages vary in shape and size.

On the seventh day, the number of macrophage colonies began to increase significantly, and cytoplasmic bridges and tissue-type junctions, histiocytes, and osteoblastoma-like cells were observed between macrophages and lymphocytes. Colonies develop from reticular cells and neutrophils.

In the second cavity, characteristic, round, smooth Staphylococcus colonies are formed, the number of which has grown over time, converged, and a continuous growth zone has been formed in the cavity.

Example 7

Solutions A and C were prepared as described in Example 1 and Solutions B as described in Example 2.

The reaction mixture of the acrylamide and N, N'-methylene-bis-acrylamide monomers 15.0:

: 0.027 w / w and the ratio of the mixture to physiological saline is 1: 7. This reaction mixture is then filled into a reactor and a polyacrylamide gel formed is formed into a 120 x 75 x 2.2 mm and 120 x 75 x 1.0 mm strip, the latter forming cavities 4 x 1.2 mm, 1.7 mm apart, in a coaxial arrangement.

The copolymerization is carried out for 40-50 minutes and the strips are soaked in a mixture of the composition of the starting monomer mixture and superimposed so that closed cavities are formed and held together until a uniform block is formed. Then we divide it into sections so that each one contains a cavity. The resulting diffusion chambers were then washed in saline and swollen at 2.8 times their weight in 0.85% saline, then sterilized for 1-1.5 hours and finally saturated with medium 199.

From the femoral bone of intact syngeneic animals, bone marrow cells (4 x 10 ⁶ ) were resuspended in 0.2 ml of medium 199 and placed in the diffusion chamber and implanted into the abdominal cavity of syngenic recipients.

After in vivo culturing of the cells, the diffusion chamber is removed from the abdominal cavity and examined by microscopic examination of the cell monolayer (growth zone, cell culture) and contact preparation from cell cultures.

Bone marrow cells cultured in the diffusion chambers of the abdominal cavity of normal animals on the third day of culture exhibited a heterogeneous cell structure characterized by intact bone marrow. On the seventh day, some of these cells degenerate, and some begin to convert to a specific form. Different steps of granulocyte differentiation can be observed. In the vicinity of fast-growing filaments of non-differentiated tissues, metamyelocyte cells form clusters and form arches on their surface. The mature granulocytes contain a nucleus that is characterized as a neutrophil. In their intermediate forms, pronounced core segmentation is observed. In the growth zone, large epithelial cells (40 pm) are formed with insignificant proliferation on their surface, become monolayer clustered and isolated, their nuclei are flattened and phagocytic inclusions are absent in the cytoplasm.

Monocytes appear to be extremely viable. Phagocyte vacuoles are often filled with content. Monocytes are identified by their bean-like shape, which is often eccentric. After a longer cultivation time, the seed is rounded. Monocytes are often located near the epithelial cells but do not show a tendency to aggregate.

In 21-day cultures, growing cells fill the cavity of the diffusion chamber and form a defined cellular organization. The layer lying directly on the surface is made up of epithelial cells, followed by a group of monozytes, adipose cells and neutrophils in young form. Mature forms gather on the periphery. Lymphocyte-like and germ cells are located within the adhesive layer. In the growth zone, fat cells appear as small, dense nuclei, often in the form of dicotyledons. It is well recognized by electromicroscopy as small droplets of fat on the cytoplasm. Lipid outbreaks often develop in nest-like form, sometimes fusing and forming droplets of 40 µm. Due to the accumulation of fat, these cells sometimes reach 120 µm. It refers to the intracellular lipid synthesis of an isolated vacuole membrane. A round and oval core consists of condensed chromatin. The cytoplasm of mature cells contains a number of mitochondria. During the previous steps of fat accumulation, the cytoplasm is neutralized. Fat cells tend to form clusters and form foci between granulocytic colonies but retain their functional dependence for a longer period of time.

-8193585

Megakaryocyte cells may also be observed during the assay. These giant cells are spherical and sometimes arched on the surface of the cytoplasmic membrane. No Trom bocyte formation was observed.

Example 8

Solution A and C were prepared as in Example 1 and Solution B as described in Example 4.

The reaction mixture contains acrylamide and N, N'-methylene-bisacrylamide monomers in a weight ratio of 10.0: 0.132 and a weight ratio of the mixture to physiological solution of 1: 5. The reaction mixture is poured into a reactor and two flat strips of the resulting polyacrylamide gel are formed. One of them is 150 x 80 x 12 mm, the other 150 x 80 x 2.5 mm, and the latter is formed 5 x 1.3 mm cavities 1.8 mm apart, in a coaxial arrangement. The copolymerization was carried out for 40 minutes. The polyacrylamide-based strips are then impregnated with a mixture corresponding to the starting monomer mixture and placed in such a manner that closed cavities are formed, and then held for 50 minutes until a uniform block is formed, and then divided into pieces so that each is closed. contain a cavity. The resulting diffusion chambers were swelled in saline 3.2 times their weight, then sterilized and impregnated with Hanks' solution.

For the cultivation of healthy human leukocytes, a leukocyte population is placed in the wells of the diffusion chamber, containing 1 x 10 ⁶ volumes of all forms of white blood cells, suspended in Hanks' solution. The diffusion chambers are then implanted into the organs of xenogeneic animals (mixture, non-purebred) and removed after the desired observation time. Evaluation of the experiments showed that the lymphocytes survived for 20 days, although they decreased from 79% to 20%. Neutrophils and monocytes remained viable, at least for the first 2-3 days. Macrophages increased to 15% on the second day and 50% on the twentieth day. On the eighth day, germ, fibroblast-like and transitional cells appeared in the culture.

Claims (6)

PATENT CLAIMS A process for the preparation of a solid culture medium for cultivation of cell cultures of microorganisms and macroorganisms in a physiological solution of acrylamide and Ν, Ν'-methylene-bis-acrylamide in the presence of a radical initiator, followed by Washing of 5 polyacrylamide gels with physiological saline, characterized by copolymerization of a (15.0—20.0): (0.019-0.132) weight ratio of acrylamide-Ν, metil-methylene-bis-acrylamide monomer. solution and, after washing, an excellent polyacrylamide gel. a) swelling with saline until its mass is increased to 2.8 to 4.5 times and then treated with a liquid medium. You are 15 b) swelling with a liquid medium until its mass is increased 2.5 to 3.5 times. 2. A process according to claim 1, wherein the copolymerization is carried out in an amount of 1: 4 to 1: 7 by weight of the acrylamide-Ν, Ν-methylene-bis-acrylamide monomer mixture. 25 3. A process according to any one of claims 1 to 5, characterized in that the saline solution is 0.85% aqueous sodium chloride, Ringer-Locke, Earl, Hanks or 5% glucose solution. 30 The method of claim 1, wherein the liquid medium is Hottinger's trypsin digestion solution, Marten's broth, peptone-aqueous medium, casein hydrolyzate, salt mixture or medium 199. 35 applied. 5. The process according to any one of claims 1 to 3, characterized in that a 1: 1 mixture of human blood serum and plasmol inactivated to swell the polyacrylamide gel is used. 40 as a liquid medium. 6. A process according to any one of claims 1 to 6, characterized in that the copolymerization is carried out by forming the polyacrylamide gel into two flat strips, one of which forms cavities in a coaxial and / or chessboard arrangement, and then strips 15.0 to 20.0: 0.019 - Impregnated with 0.132 wt.% Acrylamide-N, N'-methylene-bis-acrylamide monomer mixture 50 by forming closed cavities and forming a unitary block therewith dividing them into at least one closed cavity.