WO2018012993A1 - Method for maintaining viability of treponema pallidum culture strains via cryopreservation - Google Patents

Abstract

The invention relates to the field of microbiology. Proposed is a method for maintaining the viability of treponema pallidum culture strains via cryopreservation. The method involves: accumulating treponema biomass in a liquid nutrient medium, concentrating via a natural sedimentation method with the subsequent decanting of supernatant liquid, and stabilizing the concentrated microbial suspension using a protective sucrose/glycerol medium. The produced preparation is packaged in individual airtight sealing elements and frozen for subsequent storage at a temperature of negative 40±1°C. The use of the method guarantees the ability to maintain the viability and growth characteristics of treponema culture strains for 18 months.

Description

 METHOD FOR PRESERVING THE VITALITY OF CULTURAL STRAINS OF WALK TREPONES USING CRYO-CONSERVATION

 The invention relates to microbiology and can be used in studies of the biological properties of the causative agent of syphilis, while maintaining and maintaining the cultural strains of pale treponemas, as well as in the production technology of therapeutic and diagnostic preparations based on them.

 State of the art

The causative agent of syphilis is pale treponema (spirochete), discovered in 1905 by F. Schaudinn and E. Hoffman. According to Bergey's classification, it belongs to the Gracillicutes division, the Spirochaetales order, the Spirochaetaceae family, the Treponema genus, the Treponema pallidum species, the Treponema pallidum pallidum subspecies ( ^{^} Identifier of the Bergie bacteria. Reference book in 2 volumes / edited by J. Holet, N. Kriga , P. Snita / transl. From English under the editorship of G.A. Zavarzin. - M.: Mir, 1997. - 800 p).

 Pale treponemas isolated from pathological material from humans were almost never managed to be maintained in a pathogenic state on artificial media. However, attempts to grow pale treponema on nutrient media under anaerobic conditions led to the production of a certain number of cultured strains. These treponemal strains, called “cultured” (in contrast to the pathogenic “tissue” treponemas) are avirulent not only for humans, but also for laboratory animals sensitive to syphilis - rabbits and primates.

 Cultural treponemas are somewhat different from tissue ones in their morphological, biochemical, and pathogenic properties. However, it is especially significant that their antigenic properties are very close to the pathogenic variants of the causative agent of syphilis. The greatest number of cultured strains of pale treponemas in our country were isolated by V.M. Aristovsky and R.R.Geltzer. These Kazan (I, II, IV, V) and Stavropol (VI, VII, VIII, IX) strains along with the Reiter strain are used to prepare antigens for serodiagnosis of syphilis (Ovchinnikov, N.M. Experimental syphilis / N.M. Ovchinnikov .-- M .: Medgiz, 1955 .-- 387 s).

It should be noted that the causative agent of syphilis is extremely whimsical. microorganism. Cultural treponemas, in comparison with other heterotrophic microorganisms, are characterized by high nutritional requirements, do not grow on ordinary nutrient media, develop in a narrow temperature and acid-alkaline range, require careful observance of anaerobic conditions, are characterized by a very slow (up to 7-10 days) growth and low biomass accumulation.

 Pale treponemas develop well on the chorionallant tissue of a chicken fetus, in rabbit serum with the addition of pieces of brain tissue under a layer of paraffin oil. The most common culture media for cultivating treponemes are culture media, the nutritional basis of which is bovine heart broth with pieces of liver or bovine heart with the addition of peptone and growth factors (Yu.A. Kozlov, Culture media in medical microbiology, M.: Medgiz, 1950, pp. 203-206 .; Handbook of Microbiological and Virological Research Methods, edited by MO Birger, Moscow: Medicine, 1973, pp. 47-92). Media based on a mixture of ascitic fluid with hepatic broth (pH 7.4–7.6) were developed, in which slices of the liver were replaced by slices of agar or finely chopped, boiled chicken yolk. The use of liquid nutrient media based on protein hydrolysates of food and non-food raw materials is described (RU 2198920 C1, 02.20.2003). A dry commercial environment for obtaining biomass of treponems of Reuters strain Tremoneum Omata Bouillon for Treponemas and Fusobacteria (HiMedia Laboratories Pvt. Limited, India) is offered on the market.

 The closest to the essence and purpose of the claimed method of maintaining cultural treponemal strains is the traditional method of subculturing (Ovchinnikov, N.M. Experimental syphilis / N.M. Ovchinnikov. - M.: Medgiz, 1955. - 387 s), based on regular and frequent (1 time in 2-4 weeks) transfers in liquid nutrient media. To maintain pure cultures, liquid nutrient media based on meat and peptone broth with the addition of calf liver pieces in the amount of 0.25% of the volume of the nutrient medium with a pH of 8.0-8.4 are most often used. Cultivation is carried out at a temperature of 37 ± 1 ° C for 5-7 days. in static anaerobic conditions.

It must be borne in mind that the subculturing method is characterized by a number of significant drawbacks: the complexity, the high cost of nutrient media prepared on the basis of high-quality food raw materials, the danger of contamination the ministry of crops by extraneous microflora in the process of reseeding, the threat of changes in the basic biological properties of the pathogen. The indicated difficulties in preserving the strains lead to the need for systematic duplication of reseeding and maintaining ties with institutions that preserve the cultured strains of pale treponemes.

 It is known that low-temperature preservation is characterized by a small damaging effect and is regarded by many authors (Nikitin, E.E., Zvyagin I.V. Freezing and drying of biological preparations / E.E. Nikitin, I.V. Zvyagin. - Moscow , 1979. - 337 p .; Pushkar N.S., Belous AM Introduction to cryobiology. - Kiev: Naukova Dumka, 1975. - 175 p.) As one of the promising methods for storing microbial cultures, which today is widely used in laboratory practice. The effectiveness of this method is determined by strict observance of the storage regime and the range of temperatures used (Ashwood-Smith, M. Preservation of microorganisms by freezing, drying from a frozen state and dehydration in a desiccator / M. Ashwood-Smith / translated from English - Moscow, 1982. - 36 s). To increase the stability of microbes during low-temperature preservation and subsequent storage, it is necessary to use cryoprotective media (cryoprotectants).

 It should be noted that a similar approach is used to preserve the viability of the pathogenic strain of pale treponema (Treponema pallidum pallidum Nichols strain), which is not susceptible to cultivation on artificial nutrient media and is maintained exclusively in the body of a susceptible laboratory animal (rabbit). It includes: testicular infection of the rabbit with a culture of pathogenic pale treponema; obtaining from the testis suspension of microbes containing an extract of native tissues; suspension stabilization with a cryoprotectant (glycerin); separation into small aliquots of 0.75-1.5 ml; freezing and storage at a temperature of minus 70-80 ° С (RU 2292388 О, January 27, 2007).

 Disclosure of invention

The objective of the invention is to develop a method that preserves the viability of the cultured strains of pale treponema for a long period of time, while simplifying the method and reducing costs associated with the acquisition and / or preparation of expensive food raw materials of proper quality (nutrient media) , and eliminating the danger of contamination of crops by extraneous microflora in the process of regular reseeding. The technical result is to increase the life span of the culture strains of pale treponemas.

 The problem is solved in that the method of maintaining the viability of the cultured pale treponem strains using cryopreservation involves obtaining stabilized microbial suspensions from native concentrated cultures of treponemal strains by adding a cryoprotective medium, followed by freezing and storage of stabilized microbial suspensions at a temperature of minus 40 ± 2 " C. In a particular embodiment of the invention, the addition of a cryoprotective medium can be carried out in a volume ratio of 1: 2 (about on a part of the protective environment and two parts of the culture of microbes.) Native concentrated cultures of treponemal strains are obtained from native deep cultures using the natural precipitation method followed by removal of the supernatant. Native deep cultures of treponemal strains are obtained from seed material on a commercial Omata Bouillon for Treponemus and Fusobacteria ”, the cultivation of which is carried out in meat and peptone broth with the addition of pieces of the liver. In the inventive method, a cryopreservation medium of any composition can be used, which ensures the viability of culture treponemes during freezing and storage. In a particular embodiment of the invention, a protective medium is used comprising, wt.%: Sucrose - from 7 to 8%, glycerin - from 28 to 32%, potassium disubstituted phosphate - from 0.5 to 0.7%, water - up to 100%.

 The technical result is achieved due to the fact that biomass of treponems is accumulated in a liquid nutrient medium, concentrated by natural sedimentation followed by decantation of the supernatant, and stabilization of the concentrated microbial suspension by cryoprotective sucrose-glycerol medium. The resulting preparation is packaged in separate airtight closures, for example, 5 ml each and immediately subjected to freezing for subsequent storage at a temperature of minus 40 ± 2 ° С.

The concentration of treponem biomass ensures the creation of the required seed dose for subsequent reseeding, the addition of a cryoprotective sucrose-glycerin medium determines the protective effect during freezing and storage, packing in separate airtight closures allows achieving optimal rates of freezing and thawing of the obtained product, and subsequent storage at minus 40 ± 2 ° C ensures viable microorganisms for at least 18 months. (according to experimental studies).

 The combination of the above operations and the conditions for their implementation provides the following benefits:

 5 during the uninterrupted operation of refrigeration equipment, the viability and growth properties of the cultured strains of pale treponemes are guaranteed to be maintained for at least 18 months;

 cryopreserved cultures of treponemal strains after reactivation can be used for direct inoculation of liquid nutrient media from the calculation of 10 contents of one closure per 0.25 - 0.5 l of medium;

 monitoring at all stages of preparation of cryopreserved cultures of treponemal strains ensures the absence of extraneous microflora and a reduction in the losses associated with this observed during repeated reseeding; the periods of cultivation of treponemal strains under anaerobic conditions at a temperature of 37 = 1 = 1 ° C when using cryopreserved cultures as seed material practically do not differ from the analogous when using deep cultures as seed material (7-10 days). );

 storing cryopreserved cultures in separate airtight closures allows fractional use of the initially prepared preparation without 20 deterioration of its properties and is repeatedly used as a seed;

 the systematic application of this method reduces direct material and labor costs associated with the acquisition and preparation of nutrient media, the implementation of regular reseeding, including the temporary cessation of work with cultured strains of pale treponemes.

25 Best Mode for Carrying Out the Invention

 The method is as follows:

 receive seed material of treponemal strains by growing microbes, for example, in meat and peptone broth with the addition of 20 to 25% by volume of boiled beef liver piles in flasks under anaerobic conditions at a temperature of 30 37 ± GS for 7 to 10 day .;

get native deep cultures of treponemal strains by growing microbes, for example, in the commercial liquid nutrient medium “Omata Broth for Treponemus and Fusobacteria” (HiMedia Laboratories Pvt. Ltd., India) with the addition of commercial cattle serum in flasks under anaerobic conditions at a temperature of 37 ± C for 7 to 10 days. in a shaker-incubator at a platform speed of 120 ± 10 rpm ^"1 ;

 native concentrated suspensions of treponemal strains are obtained using the natural sedimentation method, followed by decantation of the supernatant, which includes settling the microbial culture obtained at the previous stage for a day at a temperature of 20 ± 2 ° С until a formed sediment is obtained, followed by aseptic removal of the supernatant. dosing fluid in a volume of 60 to 80% of the initial volume of the culture fluid;

 stabilized microbial suspensions of the treponemal strains are prepared by adding a cryoprotective medium comprising the following components, wt.%: sucrose - from 7 to 8%, glycerin - from 28 to 32%, potassium disubstituted phosphate - from 0.5 to 0.7% ;

 in stabilized microbial suspensions of treponemal strains, the appearance, typical properties of microbes and the absence of extraneous microflora are monitored by phase contrast microscopy in native preparations of the “crushed drop” type;

 Stabilized microbial suspensions of the treponem strains are packaged in separate sealed closures;

 within 1 hour from the preparation, stabilized microbial suspensions of treponemal strains are frozen at a temperature of minus 40 ± 2 "C; storage of cryopreserved microbial suspensions of treponemal strains is carried out at a temperature of minus 40 ± 2" for up to 18 months.

 The feasibility of the proposed method with the achievement of a technical result is confirmed by examples of specific performance.

 Example Na 1. The method of cryopreservation of culture strains of pale treponema on the example of the strain Treponema pallidum pallidum Reiter

 To obtain inoculum with treponem, a flask with a capacity of 0.5 l, filled in three quarters with sterile meat and peptone broth with pieces of liver under a cotton-gauze stopper, aseptically introduced a culture of Reiter strain in an amount of 40 to 60 ml. To create anaerobic conditions, a sterile medical petroleum jelly, previously melted in a water bath, was carefully layered onto the surface of the seeded medium, so that an airtight plug 1.5-

2.0 cm. The contents of the seeded flasks were thoroughly mixed with neat rotational movements and placed in a thermostat at a temperature of 37 ± GS for a period of 7 to 10 days. Every day several times a day, the growing cultures were mixed with neat rotational movements to evenly distribute microbes throughout the entire volume of the medium.

 To obtain a native deep culture, in a flask with a capacity of 0.5 or 1 L, filled in three quarters with a sterile commercial Reuters broth with the addition of 10% of the native cattle serum, aseptically added about 10-20% of the seed prepared on meat and peptone broth by volume. To create anaerobic conditions, a sterile medical petroleum jelly pre-melted in a water bath was carefully layered on the surface of the seeded medium, so that an airtight stopper 1.5-2.0 cm high was obtained. The contents of the seeded flasks were thoroughly mixed and placed in a shaker incubator at a temperature 37 ± C and a platform frequency of 120 ± 10 swing-min-1 for a period of 7 to 10 days. Several times a day, visual growth control was performed.

 To obtain a concentrated native culture, at the end of the cultivation, the flasks were left at room temperature for 24 hours to condense the precipitate. After this period, a vaseline plug was removed from each flask aseptically using a sterile glass spatula. Then, using a vacuum, supernatant (60 to 80%) was carefully removed from each flask in such a way as not to disturb the integrity of the precipitate. Conditioned precipitates from all flasks were aseptically uniformly combined into one sterile container. The culture was similarly concentrated 30 times. After this, the typical morphology and the presence of extraneous microflora were monitored using microscopy.

 A cryoprotective medium was added to the concentrated suspension in a volume ratio of 1: 2. The contents of the container were thoroughly mixed. After that, the typicality of the morphology of microbes and the absence of extraneous microflora in native preparations of the “crushed drop” type were monitored using phase contrast microscopy.

The stabilized microbial suspension was packaged in 5 ml in sterile 10 ml bottles, corked with rubber stoppers, which were fixed with metal caps. Packaged and labeled vials with a stable concentrated treponem culture were immediately placed in a low-temperature refrigerator with a temperature of minus 40 ± 2 ° C for freezing and storage.

 By the claimed method, 3 series of cryopreserved cultures were prepared. During storage, the safety of the viability and growth properties of microbes was regularly monitored (Table 1). For this, vials with a cryopreserved strain culture were reactivated for 30 minutes. at a temperature of 37 ± GS, and then inoculated into 0.25 L flasks containing meat and peptone broth with pieces of liver. Cultivation was carried out from 7 to 10 days. at a temperature of 37 ± C with daily stirring, macro- and microscopic control of growth.

 The inventive method was tested using the cultural strains Treponema pallidum pallidum “Kazan” V, “Stavropol” VII, “Stavropol” VIII, “Stavropol” IX (see examples 2 to 5 of table 1).

The results of the studies show that after freezing and storage of cryopreserved cultures of treponemal culture strains for 18 months. at a temperature of minus 40 ± 2 ° С, their growth properties in a liquid nutrient medium practically do not differ from similar indicators for microbes stored for 1-2 months. at room temperature in a meat-peptone broth with pieces of liver.

Table 1 - The results of assessing the viability and growth properties of cultural treponemal strains after freezing and storage in a cryopreserved state

Claims

CLAIM

1. A method for maintaining the viability of cultured pale treponemal strains using cryopreservation, comprising obtaining stabilized microbial suspensions from treponemal native concentrated cultures by adding a cryoprotective medium, followed by freezing and storage of stabilized microbial suspensions at a temperature of minus 40 ± 2 ° С.

 2. The method according to claim 1, characterized in that the addition of cryoprotective medium is carried out in a volume ratio of 1: 2.

 3. The method according to claim 1, characterized in that the native concentrated cultures of treponemal strains are obtained from native deep cultures using the method of natural deposition followed by removal of the supernatant.

 4. The method according to claim 3, characterized in that native deep cultures of treponemal strains are obtained from seed on the commercial Omata Bouillon for treponemas and fusobacteria, the cultivation of which is carried out in meat and peptone broth with the addition of pieces of liver.

 5. The method according to claim 1, characterized in that a sucrose-glycerin cryoprotective medium is used.

 6. The method according to claim 5, characterized in that the cryoprotective medium is used with the following components, wt.%: Sucrose - from 7 to 8%, glycerin - from 28 to 32%, potassium disubstituted phosphate - from 0.5 to 0 , 7%, water - the rest.