RU2542481C2 - Method of production of bacteriologically pure cultures of marine blue-green microalgae - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of production of bacteriologically pure cultures of marine blue-green algae provides chemical sterilisation of microalgae cultures by treatment them in the solution of sterile sea water containing 0.1% phenol and 1.0% ethyl alcohol. Microalgae are kept in sterile sea water containing phenol and ethyl alcohol in a predetermined ratio for 4-5 hrs. Irradiation with UV light is carried out for 9-10 minutes. The treatment of microalgae with antibiotics and fungicide is carried out. At that the antibiotics are used as chloromycetin and ampicillin in the amount of 50 µg/ml each, and fungicide is used as nystatin in the amount of 25 µg/ml.

EFFECT: invention enables to improve the efficiency of extraction of bacteriologically pure culture of blue-green microalgae.

1 tbl, 20 ex

Description

The invention relates to algology and microbiology, and in particular to methods for isolating bacteriologically pure cultures of marine blue-green microalgae.

Currently, much attention is paid to the development of test systems for assessing the toxicity of the components of ecosystems of marine water bodies, for which it is necessary to isolate pure cultures of aquatic organisms, in particular microalgae. The standard procedure is to isolate and grow algologically pure microalgae cultures, from which the most promising ones for biotesting are then selected. However, the isolated microalgae cultures always turn out to be contaminated with bacterial microflora, spores and fungal mycelium, which interferes with further research and distorts their results. Removing bacteria is a very time-consuming process, as between algae and bacteria there are dense biocenotic connections. Algae mucous membranes serve as a source of nutrition and shelters for microorganisms. Such a close relationship determines the difficulties of dividing such communities into isolated cultures: algae and bacteria. Obtaining bacteriologically pure cultures of microalgae is a prerequisite for successful research, development of test systems for assessing the toxicity of components of ecosystems of marine water bodies and other scientific works.

There is a method of isolating axenic cultures of microalgae by the method of agar plates (Vasser S.P., Kondratieva N.V., Masyuk N.P. and other algae. Reference book. - Kiev: Naukova Dumka, 1989. - 608 p.) (1) in which the microalgal suspension is introduced into a Petri dish and the microbiological loop is evenly distributed over the surface of the meat-peptone agar (MPA), followed by incubation in a thermostat. Then, individual microcolonies grown on the surface of meat-peptone agar are removed with sterile instruments together with pieces of a dense nutrient medium and introduced into test tubes with appropriate sterile elective nutrient media to accumulate algae biomass. Axenic cultures of algae are obtained by repeated re-sieving on the surface of MPA under sterile conditions.

The known method has a limited range of applications. For example, when isolating axenic cultures of marine blue-green algae, it is not possible to achieve their complete freedom from bacteria, because the latter are strongly associated with the highly developed mucous membranes of these microalgae. The process of separating them is time-consuming, time-consuming and sometimes inefficient, requires multiple reseeding.

In another method of purification of cultures from bacteria and other microorganisms, micro-algae cultures are irradiated with ultraviolet light (Kvitko K.V. Obtaining cultures of individual chlorella cells // Studies in Genetics. - L .: Publishing House of Leningrad University, 1961. - Issue 1. - S.50-54) (2). However, this method is not effective enough, because it is not possible to achieve complete freedom of algae from bacteria and fungi. In this case, the duration of ultraviolet radiation should not exceed 10 minutes, because longer UV exposure has a mutagenic effect and can cause irreversible genetic changes in the irradiated culture.

Closest to the proposed method is the selected as a prototype method of purification of cultures from bacteria and other microorganisms (in particular, fungi) by chemical sterilization by treatment in 0.1% phenol and 1.0% ethyl alcohol. (Ecological physiology of marine planktonic algae. Kiev: Publishing House "Naukova Dumka", 1971, p.5-22) (3).

However, chemical sterilization of microalgae also does not allow to obtain completely bacteriologically pure cultures of microalgae.

The inventive method solves the problem of increasing the efficiency of isolation of bacteriologically pure culture of marine blue-green microalgae.

This object is achieved in that the method for producing bacteriologically pure cultures of marine blue-green microalgae consists of chemical sterilization of microalgae cultures by treatment in a solution of sterile sea water containing 0.1% phenol and 1.0% ethyl alcohol, and additional irradiation with ultraviolet light for 9-10 min, and then treatment with antibiotics and a fungicide, moreover, as antibiotics use chloramphenicol and ampicillin in an amount of 50 μg / ml each, and as a fungicide use nis tatin in an amount of 25 μg / ml.

Additional processing of microalgae after chemical sterilization, first by irradiation with ultraviolet light, and then with antibiotics and a fungicide, allows to obtain a bacteriologically pure microalgae culture.

At the same time, the use of chloramphenicol in the amount of 50 μg / ml and ampicillin in the amount of 50 μg / ml as antibiotics, and nystatin in the amount of 25 μg / ml as the fungicide, allows, on the one hand, an almost complete absence of bacteria in the marine blue culture green microalgae, and on the other hand, avoid reducing the growth rate and inhibiting the division of microalgae.

As a result of the analysis of the prior art, an analogue was not found, characterized by features identical to all the essential features of the claimed invention, and the determination of the prototype from the identified analogues made it possible to identify a set of distinctive features that are significant in relation to the technical result.

Therefore, the claimed invention meets the condition of "novelty."

In an additional search for other solutions related to the proposed method, these distinctive features were not found. Thus, the claimed invention meets the condition of "inventive step".

The method is as follows.

Cleaning algologically pure cultures of marine blue-green microalgae from bacteria is carried out in 3 stages. Stage 1 is chemical sterilization. For this, antiseptics such as phenol and ethyl alcohol are used at a concentration of 0.1 and 1.0%, respectively. Algae are kept in sterile sea water with the above antiseptics for 4-5 hours. At the 2nd stage, a 9-10-minute irradiation of algae cultures with ultraviolet light is carried out (bactericidal irradiator OBN - 150.2 lamps of 30 W each) in Petri dishes a distance from the radiation source of 50 cm. At the 3rd stage, the algae are treated with antibiotics and a fungicide in the following optimal combination: chloramphenicol at a concentration of 50 μg / ml, ampicillin at a concentration of 50 μg / ml and nystatin at a concentration of 25 μg / ml.

The presence of bacterial microflora was controlled by reseeding from liquid cultures of marine blue-green microalgae onto agarized or liquid nutrient media. The growth of mesophilic aerobic and facultative anaerobic microorganisms, molds, and yeast was controlled.

When determining mesophilic aerobic and facultative anaerobic microorganisms in a liquid microalgae culture, the study was carried out according to GOST 10444.15-94.

When determining mesophilic aerobic and facultative anaerobic microorganisms on agarized nutrient media, 1 cubic meter. see cultures of blue-green algae were sown in two parallel Petri dishes. Crops were poured in accordance with GOST 26670 one of the nutrient agarized media and incubation at a temperature of 30 ° C.

When determining mesophilic aerobic and facultative anaerobic microorganisms in liquid nutrient media, 1 cubic meter. see cultures of blue-green algae were sown in two flasks or test tubes with one of the liquid nutrient media according to GOST 26670. The ratio between the amount of sown algae culture and the nutrient medium from 1: 5 to 1: 7. Crops were incubated at a temperature of 30 ± 1 ° C for 72 ± 3 hours under aerobic conditions.

After incubation of crops on agarized culture media in Petri dishes, the presence or absence of colonies of microorganisms was taken into account. In liquid nutrient media, the presence or absence of visible signs of growth (gas formation, the appearance of turbidity, sediment) was noted.

If there was no obvious bacterial growth after three days of incubation, it was considered unlikely that the algae sample added to the culture medium contained bacteria. However, the culture was continued to be observed for 7-10 days, as slowly growing species could be present in it. Conclusions about the purity of cultures were made in the absence of colonies of microorganisms or visible signs of their growth.

When determining molds and yeasts in a liquid microalgae culture, the study was carried out according to GOST 10.444.88.

1 cc. see algae cultures were seeded in two Petri dishes with Saburo medium with antibiotics. Crops are incubated at 24 ± 1 ⁰ C for 5 days. At the end of the incubation, the presence or absence of visible signs of growth was noted. Colonies of yeast and molds were visually separated (yeast form large convex shiny colonies with a smooth surface and even edge, molds form mycelium of various colors).

In the absence of mesophilic aerobic and facultative anaerobic microorganisms, molds, and yeast, the liquid microalgae culture was considered bacteriologically sterile.

The method is illustrated by the following examples.

Example 1 (control). Water samples and scrapings of microscopic algae from coastal stones for subsequent isolation of microalgae cultures from them were taken during the active vegetation period (May-June) of the main types of microalgae in the Sea of Azov and the Black Sea. Initially, accumulative mixed cultures of microalgae were grown, for which Bristol medium in the modifications of Gollerbach, a mixture of trace elements and soil extract in certain proportions were added to the selected samples. Cumulative microalgae cultures were obtained by seeding a mixed culture in flasks with liquid nutrient media intended for laboratory cultivation of various algae (green, nitrogen-fixing and non-nitrogen-fixing blue-green). Algae cultivation time averaged 3-4 weeks.

Algologically pure cultures were obtained from an accumulative culture using microbiological methods of reseeding and dilution. Cumulative culture was sterilely plated on a Petri dish with agar medium. Cups were placed in the light for algae growth. From a colony of grown algae, part of the culture was transferred back to the liquid medium or to test tubes with a beveled agar medium. The cultivation of microalgae was carried out on the nutrient media of Tamiya, Yaguzhinsky, Guseva and Walna. The media are universal for all types of algae and make it possible to obtain shared microalgae cultures that are physiologically active. For the separation of microalgae used a glass spatula and sieving by rubbing into the surface of the nutrient medium. This allows you to break down a mixture of microalgae into single cells, which preserves their integrity, and therefore, significantly facilitates and accelerates the process of isolation of pure cultures.

As a result of the work, algologically pure cultures of the following blue-green microalgae were isolated: Oscillatoria laetevirens, Spirulina tenussima and Snowella rosea. Next, each of the obtained microalgae cultures was purified from bacteria by chemical sterilization (1). For this purpose, antiseptics such as phenol and ethyl alcohol were used at a concentration of 0.1 and 1.0%, respectively. Algae were kept in sterile sea water with the above antiseptics for 4-5 hours (table 1, experiment 1).

The purity of the obtained algal cultures was checked by control sowing of their liquid cultures on that of the above agarized culture media, on which the best growth of this culture was observed. At the same time, microflora growth was recorded. Thus, chemical sterilization did not allow to obtain absolutely pure cultures of algae. The data are shown in table 1, experiment 1.

Table 1 The growth of microflora in cultures of marine blue-green microalgae in experiments with different sterilization methods No. Chem. sterilization UV
irradiation Antibiotics, mcg / ml Fungicide,
nystatin, mcg / ml Bacteria-
flax and mushroom growth chloramphenicol tetracycline erythromycin ampicillin one + - - - - - - + 2 + + - - - - - + 3 + + fifty - - - - + four + + fifty fifty - - - + 5 + + fifty fifty fifty - - + 6 + + fifty fifty fifty fifty - + 7 + + - - - - 25 + 8 - - fifty fifty fifty fifty 25 + 9 + - fifty fifty fifty fifty 25 + 10 - + fifty fifty fifty fifty 25 + eleven + + fifty fifty - - 25 + 12 + + - fifty fifty - 25 + 13 + + - fifty - fifty 25 + fourteen + + fifty - - fifty 25 - fifteen + + fifty fifty - fifty 25 - 16 + + fifty fifty fifty fifty 25 - 17 + + 90 fifty fifty fifty 25 - eighteen + + fifty fifty fifty 90 25 - 19 + + 90 fifty fifty 90 25 - twenty + + 90 90 fifty 90 25 -

Example 2. The cultures of the isolated blue-green microalgae were first subjected to chemical sterilization (the procedure described above), and then ten-minute irradiation of the algae cultures with ultraviolet light (bactericidal irradiator OBN-150) was carried out in Petri dishes at a distance of 50 cm from the radiation source (experiment 2 ) The purity of the microalgae cultures obtained after chemical sterilization and ultraviolet irradiation was checked by control cultures of liquid cultures on the above agarized nutrient media. At the same time, microflora growth was recorded. The data are shown in table 1, experiment 2.

Thus, chemical sterilization and irradiation of microalgae cultures with ultraviolet light did not allow bacteriologically pure microalgae cultures to be obtained.

Example 3. The cultures of the isolated marine blue-green microalgae were first subjected to chemical sterilization, then a ten-minute irradiation of the algae cultures with ultraviolet light was carried out as described in examples 1-2, and then they were subjected to antibiotic treatment with chloramphenicol at a concentration of 50 μg / ml in an appropriate nutrient liquid medium. The results are shown in table 1, experiment 3.

Examples 4-20. The experiments were carried out similarly to those described above, i.e. cultures of the isolated microalgae were first subjected to chemical sterilization, then a ten-minute irradiation of the algae cultures with ultraviolet light was carried out, and then they were treated with various concentrations of antibiotics and fungicide in the corresponding nutrient liquid medium. The results are shown in table 1, experiments 4-20.

Different concentrations and different types of antibiotics and fungicides were tested (table 1). After each treatment, the purity of the obtained algal cultures was checked by control cultures from liquid cultures on an agarized nutrient medium. Thus, antibiotics and fungicide were selected, as well as their concentrations, after the treatment of which, microflora growth was not recorded during control crops on agarized nutrient media. Antibiotics were used - chloramphenicol (50 and 90 μg / ml), tetracycline (50 and 90 μg / ml), erythromycin (50 μg / ml), ampicillin (50 and 90 μg / ml), and fungicide - nystatin in an amount of 25 μg / ml In addition, experiments were conducted with a series of samples of marine blue-green microalgae treated with antibiotics and a fungicide, but not previously subjected to chemical sterilization and / or treatment with ultraviolet light (experiments 8-10). Only treatment with chloramphenicol at a concentration of 50 μg / ml in combination with ampicillin 50 μg / ml and nystatin 25 μg / ml, performed after chemical sterilization and irradiation of the cultures with ultraviolet light, allowed us to achieve almost complete absence of bacterial growth during several transfers.

 Higher concentrations of antibiotics used were tested, which made it possible to achieve a 100% absence of bacterial microflora, but such concentrations reduced the growth rate and inhibited the division of microalgae. The research results are shown in table 1.

From the table it follows that the effect associated with the complete release of marine blue-green microalgae from concomitant microflora while maintaining the activity of microalgae cells is achieved by performing chemical sterilization, irradiating the cultures with ultraviolet light, and then treating the microalgae with antibiotics and a fungicide with the following minimum ratio of components: chloramphenicol 50 μg / ml, ampicillin 50 μg / ml, as well as fungicide, nystatin in an amount of 25 μg / ml.

Thus, a phased method for the chemical sterilization of an algologically pure culture of marine blue-green microalgae with ultraviolet radiation and the further addition of certain concentrations and compositions of antibiotics and fungicides made it possible to obtain bacteriologically pure cultures of microalgae that can be used in physiological, biochemical, cytological and genetic studies microalgae and other scientific studies. In particular, bacteriologically pure cultures of marine blue-green microalgae can be used to develop test systems based on the fluorescence of microalgae by assessing the toxicity of components of ecosystems of marine water bodies.

Using the proposed method can improve the efficiency of isolation of bacteriologically pure cultures of microalgae. This method is reliable because allows to obtain bacteriologically pure cultures of marine blue-green microalgae, regardless of the source of isolation and the degree of their bacterial contamination.

Claims (1)

A method of obtaining bacteriologically pure cultures of marine blue-green microalgae, including chemical sterilization of microalgae cultures by treatment in a solution of sterile sea water containing 0.1% phenol and 1.0% ethyl alcohol, characterized in that it is additionally irradiated with ultraviolet light for 9 -10 min, and then treatment with antibiotics and a fungicide, moreover, chloramphenicol and ampicillin are used as antibiotics in an amount of 50 μg / ml each, and nystatin is used as a fungicide stve 25 ug / ml.