RU2600883C1 - Method for maintaining viability of culture strains of pale treponema using cryopreservation - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: method provides accumulation of biomass of pale treponema in liquid accumulation nutrient medium. Method comprises concentration by natural sedimentation followed by decanting supernatant. Method then includes stabilisation of concentrated microbial suspension with protective saccharose-glycerin medium to produce a preparation. Produced preparation is packed into separate sealed containers and frozen at minus 40±2 °C with subsequent storage at same temperature.

EFFECT: invention enables to maintain viability and growth properties of culture strains of treponemas for 18 months.

4 cl, 1 tbl, 1 ex

Description

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

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 species Treponema pallidum, the subspecies Treponema pallidum pallidum (Identifier of the bacteria Bergey. Reference book in 2 volumes / Edited by J. Holt, 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 almost never succeeded in maintaining a pathogenic state on artificial media. However, attempts to grow pale treponema on nutrient media under anaerobic conditions have led to a certain number of cultured strains. The indicated treponemal strains, called “cultural” (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 identified 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 p.).

It should be noted that the causative agent of syphilis is an 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-base range, require careful observance of anaerobic conditions, are characterized by very slow (up to 7-10 days) growth and low accumulation biomass.

Pale treponemas develop well on the chorionallantoic tissue of the chicken fetus in rabbit serum with the addition of pieces of brain tissue under a layer of paraffin oil. The most common nutrient media for growing culture treponemes are environments whose nutrient basis is a bovine heart broth with pieces of liver or bovine heart supplemented with peptone and growth factors (Yu.A. Kozlov. Nutrient media in medical microbiology, Moscow: Medgiz, 1950 , p. 203-206; Handbook of microbiological and virological research methods edited by M.O. Birger, M .: Medicine, 1973, p. 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 medium for obtaining biomass of treponems of Reuters strain Trematon Omata Broth 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 culture of treponemal strains is the traditional method of subcultivation (Ovchinnikov N.M. Experimental syphilis / N.M. Ovchinnikov. - M.: Medgiz, 1955. - 387 p.), 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-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 36-38 ° C for 5-7 days under static anaerobic conditions.

It should be borne in mind that the subcultivation 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 of crops by extraneous microflora in the process of reseeding, and the threat of changing the main biological properties of the pathogen. The indicated difficulties in preserving the strains lead to the need for a systematic duplication of reseeding and maintaining links with institutions that preserve the cultured strains of pale treponema.

It is known that low-temperature preservation has 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. - M., 1979. - 337 pp .; Pushkar N.S., Belous AM Introduction to cryobiology. - Kiev: Naukova Dumka, 1975. - 175 pp.) 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 / transl. From English - M., 1982. - 36 sec.). To increase the stability of microbes in the process of 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 maintain the viability of the pathogenic strain of pale treponema (Treponema pallidum pallidum Nichols strain), which cannot be grown 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 the 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 ° C (RU 2292388 C1, 01/27/2007).

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 risk of contamination of crops by extraneous microflora in the process of regular reseeding.

The technical result consists in increasing the time for maintaining the vitality of the cultured strains of pale treponemal.

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 (38-42) ° C. In a particular embodiment of the invention, the addition of a cryoprotective medium can be carried out in a volume ratio of 2: 1. 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 on the commercial Omata Bouillon for Treponemus and Fusobacteria, which is grown in meat and peptone broth with the addition of pieces of liver. In the inventive method, a cryopreservation medium of any composition can be used that ensures the viability of culture treponemas 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.

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 with cryoprotective sucrose-glycerol medium. The resulting preparation is packaged in separate sealed closures, for example 5 ml, and immediately subjected to freezing for subsequent storage at a temperature of minus 40 ± 2 ° C.

The concentration of treponem biomass provides the necessary 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 a temperature of minus 40 ± 2 ° C ensures the preservation of the viability of microorganisms for at least 18 months (according to experimental studies).

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

during the uninterrupted operation of refrigeration equipment, the viability and growth properties of the cultured strains of pale treponemas 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 based on the 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 ° 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);

storage of cryopreserved cultures in separate airtight closures allows fractional use of the initially prepared preparation without 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 culture media, the implementation of regular reseeding, including the temporary cessation of work with cultured strains of pale treponemes.

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 pieces of boiled beef liver in flasks under anaerobic conditions at a temperature of (37 ± 1) ° C for 7 to 10 days;

native deep cultures of treponemal strains are obtained 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 ( 37 ± 1) ° 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 in the previous stage for 24 hours at a temperature of (20 ± 2) ° C until a formed sediment is obtained, followed by supernatant removal under aseptic conditions in a volume of from 60 to 80% of the initial volume of the culture fluid;

stabilized microbial suspensions of strains of treponem are prepared by adding a cryoprotective medium, including 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;

packing of stabilized microbial suspensions of treponemal strains into individual sealed closures;

within 1 hour from the time of preparation, stabilized microbial suspensions of treponemal strains are frozen at a temperature of minus 40 ± 2 ° C; storage of cryopreserved microbial suspension of treponemal strains is carried out at a temperature of minus 40 ± 2 ° C 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 1

The method of cryopreservation of culture strains of pale treponema on the example of the strain Treponema pallidum pallidum Reuters

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 plug, was added aseptically a culture of Reiter strain in an amount of from 40 to 60 ml. 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 to obtain a sealed stopper 1.5-2.0 cm high. The contents of the seeded flasks were thoroughly mixed with gentle rotational movements and placed in a thermostat at a temperature of (37 ± 1) ° C 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 depth culture, a flask of 0.5 or 1 L capacity, filled in three quarters with sterile commercial Reuters broth with 10% native cattle serum, was added aseptically with the seed material prepared in the meat and peptone broth in an amount of about 10-20% by volume . To create anaerobic conditions, sterile medical petroleum jelly pre-melted in a water bath was carefully layered on the surface of the seeded medium, so that a 1.5-2.0 cm high sealed cork was obtained.The contents of the seeded flasks were thoroughly mixed and placed in a shaker incubator at a temperature of (37 ± 1 ) ° C and a platform frequency of 120 ± 10 swings · min ^-1 for a period of 7-10 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 h 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 from each flask very carefully removed the supernatant (from 60 to 80%) so as not to violate 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 2: 1. The contents of the container were thoroughly mixed. After that, the typicality of the morphology of microbes was monitored and the absence of extraneous microflora in native preparations of the “crushed drop” type 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 stabilized concentrated treponem culture were immediately placed in a low-temperature refrigerator with a temperature of minus 40 ± 2 ° С for freezing and storage.

The inventive method was prepared three series of cryopreserved cultures. 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 min at a temperature of (37 ± 1) ° С, and then they were inoculated into 0.25 L flasks containing meat-peptone broth with pieces of liver. Cultivation was carried out from 7 to 10 days at a temperature of (37 ± 1) ° С with daily stirring, macro- and microscopic control of growth.

The inventive method was tested using cultural strains Treponema pallidum pallidum "Kazan" V, "Stavropol" VII, "Stavropol" VIII, "Stavropol" IX (see examples 2-5 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 meat and peptone broth with pieces of liver.

Claims (4)

1. A method for maintaining the viability of cultured pale treponemal strains using cryopreservation, comprising obtaining stabilized microbial suspensions from native concentrated cultures of treponemal strains by adding sucrose-glycerol cryoprotective medium, followed by freezing and storage of stabilized microbial suspensions at a temperature of minus 40 ± 2 ° С. 2. The method according to p. 1, characterized in that the addition of cryoprotective medium is carried out in a volume ratio of 2: 1. 3. The method according to p. 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 p. 3, characterized in that the native deep cultures of treponemal strains are obtained from seed on the commercial Omata Broth for treponemas and fusobacteria, the cultivation of which is carried out in meat and peptone broth with the addition of pieces of liver.