Method for proximate determination of non-sterility of broth for microorganism, animal cell and virus cultivation in bioreactors
FIELD: biotechnology, in particular biopreparation production.
SUBSTANCE: claimed method includes feeding of sterilized broth into presterilized inoculator or bioreactor equipped with means for redox-potential (eH) controlling, including eH electrode and microprocessor unit for controlling and adjustment of eH and pH measurement of redox potential value for 1 h under stirring and comparison of steady-state eH values with steady-state values. When redox-potential value deviates from steady-state value of broth redox-potential by 10 % said broth is recognized as non-sterile one.
EFFECT: process of decreased cost.
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The invention relates to biotechnology and can be used in research and in the production of Biologicals (vaccines, diagnostics and other biologically active substances) by the method of deep cultivation.
Known microbiological methods for determining the open nutrient media for cultivation of microorganisms, animal cells and viruses through seed samples on meat-peptone broth (BCH), meat-peptone agar (MPA) or agar Saburo, then leave several test vials with medium at 37°3-4 days, and then at room temperature for 10-12 days (Handbook of Veterinary drugs" edited Drawsize. - M.: Kolos, 1981 P.47.) the prototype.
However, this method is time-consuming and not allow a rapid (1 hour) to give a qualitative assessment of the sterile nutrient medium used in the preparation of inoculum (insulinopenia) and for cultivation in bioreactors.
It is known that the reaction of the metabolism of microorganisms and animal cells are the reactions of the redox type, which are characterized by the transfer of electrons from the reductant to the oxidant. Each nutrient medium for submerged cultivation of microorganisms and animal cells, depending on its composition, is particularly the e initial constant value of redox potential (eh) in the range from -1200 to +1200 mV, which can be measured using a redox electrode. Electrode by design, similar to the electrode for measuring the concentration of hydrogen ions (pH), except that its sensitive element is made of metal (platinum, gold or silver) or glass with electronic conductivity. The reference electrode, as in the measurement of pH is silver chloride electrode.
The aim of the invention is to develop a qualitative method of rapid determination of open culture media for submerged cultivation of microorganisms, animal cells and viruses in the manufacture or transfer of nutrient media in the dry inoculant and bioreactor for 1 hour before inoculation of the culture.
This goal is achieved by the fact that the definition of open media spend by changing the value of the redox potential (eh) from established within 1 hour of constant values Yong to the culture medium used before the processes of insulinopenia and cultivation of microorganisms, animal cells and viruses, which reduces the cost of production of biological products by eliminating non-sterile operations processes submerged culture.
The method is as follows. The inoculators and bioreactors for globinopathiae microorganisms, animal cells and viruses must be equipped with technical means of control of redox potential (eh), including electrode Yong and analytical microprocessor control unit and regulating the pH and eh. Sterilized nutrient medium (thermal or filter sterilization) is passed into a pre-sterilized the dry inoculant or bioreactor and produce measurement values Yong this environment (in MW) under stirring.
If within 1 hour no observed change in the value of the yen from the steady-state value, it is possible with probability P=0,9 to say that sterile nutrient medium, and the deviation values Yong from the steady-state value within more than 10% indicates kontaminirana by other organisms culture media, which will be re-sterilized in order to avoid open in the preparation of inoculum and cultivation.
As the control take at least 8 vials or test tubes (a statistically significant sample) with the sterilized environment of inoculator and bioreactor, which are sown on meat-peptone agar and leave for 3-4 days in an incubator at 37±0,5°and then conduct a visual or microscopic analysis of gains contaminating culture, which should confirm the rapid analysis of the availability of open software is teniu Yong from the original initial value for this nutrient medium
Example 1. For the cultivation of vaccine Brucella abortus strain 19 in bioreactors in the production of protivopoloznoj vaccines use a nutrient medium on the basis of parivara of Hottinger.
Ready sterilized nutrient medium in the bioreactor at a temperature of 127±1°and the pressure of 0.08-0.09 MPa for 40 min and then cooled to a temperature of cultivation (37±0,5°). For the biological control sample prepared nutrient medium were seeded on MPA or MPB and incubated at 37-38°C for 3-4 days.
Steady-state value of the redox potential of the nutrient medium should be in the range of +300 to +400 mV with an accuracy of ±10%. In the presence of contamination by other organisms within 1 hour deviation from the steady-state value is not less than ±50+60 mV. In the control samples in the case of open by deviation values Yong detected microscopically were indefitsirate culture Escerichia coli and Pseudomonas aeuruginosa 50/CFU in 1 ml of the determination Results of the open culture media for the cultivation of Brucella abortus 19 in the production of vaccines against brucellosis in cattle is shown in table 1.
|The results of the quick definition of sterility p the test environment for deep cultivation of Brucella abortus St in the bioreactor|
|No. n/n||The type and capacity of the bioreactor||Nutrient medium||Sterilization of the nutrient medium||The initial value Yong environment at 37° (MB)||The value of the yen after 1 hour (MB)||The presence of extraneous microflora in the control after 4 days|
|1||Bear-0,1 (100 l), Kirishi, Russia||Based parivara of Hottinger with additives||In the bioreactor at (127±1° (C) within 10 min||+350±10%||+280±10%||Escerichia coliu Pseudomonas aeuruginosa 50 CFU in 1 ml|
|2||Bioflo-6000 (180 l) "NBS" USA||"-"||In the bioreactor at 127±1°C for 40 min||+400±10%||+396±10%||No|
From table 1 it is evident that nedostatochna sterilization of the nutrient medium in the bioreactor BIOR-01 resulted in the open, as evidenced by changing the initial value Yong nutrient medium in the bioreactor without aeration with stirring for one hour from +350 to +280 mV. This was confirmed by 8 control samples over 4 days (contamination nutrient cultures Escerichia coli and Pseudomonas aeuruginosa). The sterility of the bioreactor Bioflo-6000 was confirmed by the fact that the value Yong nutrient medium was not changed during the Asa within the accuracy of izmereniya Yong (± 10%). The same was confirmed by the control sowing on MPA or MPB.
Example 2. A serious problem is the cleaning of the air fed to the bioreactors for aeration, suspended therein solid and liquid aerosol particles and, particularly, from microorganisms. 1 m3air contains up to 104of microorganisms. In bioreactors for the aeration of the culture medium is supplied from a 1 to 10 volumes of air per volume of medium per hour (for cultures of animal cells with surface aeration) to about 120/about·h-1(for microorganisms in bubble aeration in the bioreactor with a mixer). The cultivation cycle lasts from 4 to 200 h (bacteria and fungi) and up to 360 h (cells of animals and higher plants). The air inlet to the bioreactor and the dry inoculant must be sterile. The required degree of purification of the air supplied to the aeration after the fine filter (a filter), should be 99,9999999%. The results of determining the sterility of nutrient media for cultivation of Pasteurella multocida pieces A-1231 in the production of a vaccine against pasteurellosis pigs, insufficient cleaning and sterilization of the air supplied to the aeration in the bioreactor are shown in table 2.
Nutrient medium was prepared on the basis of the hydrolysate meat. The obtained culture medium was sterilized in the fermenter at a temperature of 127±1°and the pressure of 0.08-0.09 MPa for 40 m is N. Table 2 shows that the lack of cleaning the air supplied to the aeration in the bioreactor AK-210 caused by the lack of individual air filter, when exposed to the culture medium for 30 minutes leads to its contamination by other organisms, as evidenced by the change in yen +240 to +160 ±10% MW for one hour with vigorous stirring, which was confirmed by a control sample on the 4th day of exposure.
|The results of the quick definition of sterile nutrient medium for submerged culture of Pasteurella multocida pieces A-1231|
|No. n/ n||The type and capacity of the bioreactor||Nutrient medium||Sterilization of the nutrient medium||Sterilization of air for aeration||The initial value Yong environment at 37° (MB)||The value of EN in 1 hour||The presence of extraneous microflora in the control after 4 days|
|1||Bear-0,2 (250 l), Kirishi, Russia||Based hydrolysate meat with additives||In the bioreactor at 127°C for 40 min||Individual titanium membrane filter||+200±10%||+195±10%||No||2||AK-210 (10 l) Inst. for biological instrumentation, Pushchino on the Oka||"-"||In the bioreactor and in the autoclave at 127°C for 40min||Aeration of the nutrient medium within 30 min when removing individual filter||+240±10%||+160±10%||Eserichia coli You. B. megaterium You. stearother mophilis. 70 CFU / ml|
Example 3. For submerged cultivation in suspension transplantable kidney cells Syrian hamster (KSS-21) in the production of FMD vaccines used standard synthetic minimal medium Needle. Membrane sterilizing filtration synthetic nutrient medium when the flow in the bioreactor was carried out on the installation of filtration UPB using membranes "Millipor" or "Vladipor" with a pore size of from 0.2 to 0.3 μm. The results of determining the sterility of synthetic nutrient medium for culturing animal cells are shown in table 3.
From table 3 it follows that the puncture or rupture and sterilizing filtration membrane leads to contamination of the nutrient medium, as evidenced by the control of microbiological samples.
|The results of the quick definition of sterility synthetic grass is th environment for culturing cells KSS-21 in the production of FMD vaccines|
|No. n/n||The type and capacity of the bioreactor||Nutrient medium||Sterilization of the nutrient medium||The initial value Yong environment at 37° (MB)||The value of EN in 1 hour||The presence of extraneous microflora in control|
|1||Celly-Gen (5 l) "NBS" USA||Minimal medium Needle||Sterilizing filtration on the installation of filtration with membrane filters Millipor" or "Vladipor"||-50±10%||-49±10%||No|
|2||CC (3.5 l) "Belico Biotechnology, USA||"-"||Puncture membrane||-50±10%||+15±10%||Eserichia coli You. subtilis 50 CFU per ml|
Example 4. For submerged cultivation of primary cells of quail embryos at microsites "Cytodex-3" (pseudocumene) in the production of VirusWall against Marek's disease of chickens were used synthetic medium 199, which was sterilized through membrane filters Millipore"and microsocial sterilized in an autoclave together with a bioreactor filled with distilled water. When microneedle only opolaskivanii distilled water, but not sterilized and placed in a nutrient medium, was observed from the change of the value of the yen within 1 hour from +135 to +160± 10% mV, indicating contamination of the nutrient medium. The same was confirmed by microbiological control (see table 4).
|The results of the quick definition of sterility synthetic nutrient medium for culturing primary cells of quail embryos at microsites in the production of VirusWall against Marek's disease of chickens|
|No. n/n||The type and capacity of the bioreactor||Pete on Wednesday||Micro-media||Sterilization of the nutrient medium||Sterilization of air for aeration||The initial value Yong environment at 37° (MB)||The value of EN in 1 hour||The presence of extraneous microflora in the control after 4 days|
|1||Bioflo 111 (3,3 l) "NBS" USA||Medium 199||Cytodex-3||Sterilizing filtration using membranes "Millipor"||In conjunction with a bioreactor filled distiller bathtub with water in an autoclave at T=127°±1 for 30 min||+120±10%||+125±10%||No|
|2||"-"||"-"||"-"||"-"||Micronesia who do not sterilize them||+135±10%||+160±10%||Eserich ia coli You. B. megaterium 150 CFU per ml|
Thus, using control changes the original value of the redox potential is confirmed by the possibility of a rapid determination of the open culture media used for cultivation of microorganisms, animal cells and viruses in all cases, their possible contamination by other organisms.
The way the Express definition of open culture media for submerged cultivation of microorganisms, animal cells and viruses in bioreactors, providing for the supply of sterilized nutrient medium in a pre-sterilized the dry inoculant or bioreactor, equipped with technical means of control of redox potential (eh), including electrode Yong and analytical microprocessor unit control and regulation of eh and pH, the measurement values of the redox potential of the nutrient medium under stirring for 1 hour, and comparing values Yong-level steady-state values, and the conclusion of the open nutrient medium do when the deviation value of the redox potential of the steady-state values of the oxidation-reduction potential of pittel the Noah environment for more than 10%.
SUBSTANCE: substrate for isolation or culturing of microorganisms represents autoclaved suspension of mammalian feces with suspended particle size not more 2 mm, contains 10-7-10 g of feces per 1 l of suspension and has pH 6.0-7.6. Substrate optionally contains target additive to produce liquid or semi-liquid, or dense nutrient medium in amount of 5-300 g/l.
EFFECT: effective method for isolation and culturing of microorganism due to stimulation or selective dose-dependent inhibition of microorganism growth; decreases feces concentration; simplified method for production of claimed substrate.
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FIELD: biotechnology, microbiology.
SUBSTANCE: invention proposes a nutrient medium comprising solid and liquid phases. A solid phase represents coagulated serum, and a liquid phase comprises the nutrient medium 199, benzylpenicillin sodium salt and streptomycin solution. Invention provides enhancing the sensitivity of nutrient medium for isolation of trichomonads. Invention can be used in cultural diagnosis of trichomoniasis.
EFFECT: improved and valuable properties of medium.
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