JP4210674B2 - Mycoplasma gallicepticum culture medium - Google Patents

Description

  The present invention relates to a culture medium for culturing Mycoplasma gallicepticum whose protein source is a plant-derived protein. The medium is useful for pure culture of Mycoplasma gallisepticum and counting the number of viable bacteria (counting the number of settlements).

The medium for culturing Mycoplasma garicepticum that infects poultry and other birds has conventionally been used to grow Mycoplasma garcepticum in peptone water containing animal-derived raw materials such as animal meat extract, chicken bouillon, and milk-derived casein as protein sources. As an additive containing essential serum components, a liquid medium or an agar solidified medium to which animal serum, yeast extract, salts and saccharides are added has been used (for example, see Non-Patent Document 1). These animal meat extracts, chicken bouillon, milk-derived casein and the like, which have been added as a protein source to the medium for culturing Mycoplasma gallicepticum, are substances that have been leached or extracted from animal products or organs. The cultured mycoplasma gallicepticum is used not only to produce vaccines for preventing and reducing the incidence of birds by infecting birds such as poultry, but also as diagnostic agents for humans and animals. Also used.
Ministry of Agriculture and Forestry Livestock Hygiene Test Site 1 Supervision "Technical Guide 6 Respiratory Microplasma Disease Reproduction of Chicken" (January 30, 1972) Japan Veterinary Medical Association p. 50-53

  However, in recent years, a bovine spongiform disease (BSE) pathogen that has been transmitted to humans, particularly from ruminants, has been discovered. And the vaccine manufactured from Mycoplasma galicepticam cultured using animal raw materials containing BSE pathogens as a medium has a risk of BSE pathogen contamination, and if it is inoculated as a vaccine to poultry, meat and eggs, etc. There is a risk of human consumption and exposure to pathogens through the products of In addition, animal-derived materials generally have a high risk of transmission of pathogenic microorganisms and material contamination.

  Therefore, the object of the present invention is to avoid the use of animal-derived materials as the protein source of the culture medium for Mycoplasma gallicepticum, thereby avoiding the propagation of pathogenic microorganisms, contamination of substances, and BSE pathogen contamination caused by the use of animal-derived materials. Thus, it eliminates the risk of human consumption and exposure to pathogens through products such as meat and eggs.

  The present inventors can replace the protein present in the culture medium for mycoplasma gallicepticum as a protein source with the protein derived from plant material from the protein derived from the conventional animal material, but the mycoplasma galicepticum is better than the conventional one. As a result, the present invention has been completed. That is, the present invention relates to a mycoplasma gallicepticum culture medium, wherein the protein source is a plant-derived protein.

  There are various types of plant material-derived protein materials used in the present invention, and beans such as soybeans, broad beans, and peas are particularly preferred.

  Hereinafter, specific embodiments of the present invention will be described.

  "Polypeptone N" (manufactured by Nihon Pharmaceutical Co., Ltd.) in which purified non-heated chicken serum is 1 to 20% by volume and purified soybeans are decomposed with microorganism-derived enzyme in the purified water and / or "Polypeptone NS" in which defatted soybean is decomposed with microorganism-derived enzyme (Nippon Pharmaceutical Co., Ltd.) 0.1 to 10% by weight in total, powdered yeast extract 0.1 to 10% by weight, sodium chloride 0.1 to 10% by weight, glucose 0.1 to 10% by weight Then add sodium hydroxide to it to adjust it from weakly acidic to weakly alkaline (pH 6.0 to 8.0), and then add agar to the liquid after filtration or autoclave sterilization. The solidified material is used as a culture medium for mycoplasma gallicepticum culture. In the present invention, weight% represents w / v%.

1. Comparison of growth in liquid media <Materials and methods>
The medium of the present invention or a medium supplemented with “polypeptone” (produced by Nippon Pharmaceutical Co., Ltd.) obtained by enzymatic degradation of milk casein-derived protein instead of “polypeptone N” of the present invention medium (hereinafter referred to as “animal raw material medium”) ) And 20 vol% horse serum modified PPLO medium (25.0 g / 1,000 mL Heart Infusion bouillon containing bovine heart exudate, 0.5% by weight of powdered yeast extract, 0.5% of glucose %, Hereinafter referred to as “PPLO medium”), and 10 wt% sodium hydroxide added to adjust the pH to 7.3 ± 0.1 and pH 7.8 ± 0.1 respectively. It was. In addition, the phrase “modified method” was added because, in comparison with the medium of the present invention, glucose was 0.1% by weight in normal horse serum-added PPLO medium, but glucose was 0.5% by weight. It is.

  Mycoplasma gallisepticum 1RF inoculum was inoculated into the medium of the present invention adjusted to pH 7.3 ± 0.1.

  The original culture solution was prepared by stationary culture at 37 ° C. for 24 hours. 1/100 amount of the original culture solution was inoculated into each test medium.

  The culture solution was collected at 16 and 24 hours after inoculation, and every 12 hours thereafter, and the number of viable bacteria was measured.

The specific operation is as follows.
A two-fold concentrated liquid medium of the medium of the present invention sterilized by filtration with a 0.2 μm filter was prepared. This medium and 2% by weight Bacto Agar separately sterilized at 121 ± 2 ° C. were each kept in a constant temperature bath at 45-50 ° C., and then mixed in equal amounts, and the mixture was sterilized with plastic disposable petri having a diameter of 60 mm. 5 mL was poured into the dish and solidified. After solidification, the lid was opened in a clean bench, and it was tumbled for 1 hour and dried to obtain a viable count agar medium.

The collected culture solution was diluted 10-fold with the liquid medium of the present invention to 10 ^−5, and 10 μL of each dilution was divided into 5 drops and inoculated by dropping onto a viable count agar medium. Two groups were prepared for each step dilution, and after the inoculated liquid was absorbed into the agar, the Petri dish was turned over and stored in a 5% by volume carbon dioxide incubator. On the 10th day after storage, the number of colonies of 5 drops per group at a dilution ratio that formed 10 to 100 colonies per drop under a stereomicroscope was measured and totaled to determine the number of colonies per 10 μL. The number of colonies in the other group at the same dilution ratio was determined in the same manner, and the average of the number of colonies in the second group was defined as the number of viable bacteria.

<Result>
Tables 1 and 2 and FIGS. 1 and 2 show the results of the viable count. Table 1 and FIG. 1 are tables and drawings comparing the growth properties of the culture medium of the present invention, animal raw material medium and PPLO medium at pH 7.3, and Table 2 and FIG. It is the table | surface and drawing which compared the growth property in pH7.8.

When cultured in the medium of the present invention at pH 7.3, the maximum viable cell count was 4.5 × 10 ⁸ cfu / mL 36 hours after inoculation. It was 1.9 × 10 ⁸ cfu / mL at 60 hours for animal raw media and 1.1 × 10 ⁹ cfu / mL at 72 hours for PPLO media.

When cultured in the medium of the present invention at pH 7.8, the maximum viable cell count was 9.3 × 10 ⁸ cfu / mL 48 hours after inoculation. It was 3.1 × 10 ⁸ cfu / mL at 60 hours for animal raw media and 2.1 × 10 ⁹ cfu / mL at 96 hours for PPLO media.

  In the medium of the present invention, rapid growth was observed at any pH compared to the animal raw material and the PPLO medium. Although the maximum viable cell count did not reach the PPLO medium, it was observed that the cells grew well.

2. Comparison of detection sensitivity in agar media <Materials and methods>
In order to obtain an agar medium, first, a double-concentrated liquid medium of each of the medium of the present invention, animal material medium and PPLO medium sterilized by filtration with a 0.2 μm filter was prepared. This medium and 2% by weight Bacto Agar separately sterilized at 121 ± 2 ° C. were kept at 45-50 ° C. in a thermostatic bath, and then mixed in equal amounts. The mixed solution was poured into a plastic disposable sterilized Petri dish having a diameter of 60 mm and solidified, and then the lid was opened in a clean bench, turned over, and dried for 1 hour to obtain a viable count agar medium.

  Mycoplasma gallisepticum 1RF inoculum was inoculated into the culture medium of the present invention adjusted to pH 7.3 ± 0.2, and statically cultured at 37 ° C. for 24 hours to prepare an original culture bacterial solution. 1/100 volume of the original culture solution was inoculated into the medium of the present invention, and cultured at 16 hours, every 4 hours thereafter until 28 hours, then at 36 hours, and every 6 hours thereafter until 48 hours The liquid was used as a sample.

Samples were diluted 10-fold in steps with the liquid medium of the present invention to 10 ^−5, and 10 μL of each dilution was divided into 5 drops and inoculated on each agar medium. Two groups were prepared for each step dilution, and after the inoculated liquid was absorbed into the agar, the Petri dish was turned over and stored in a 5% by volume carbon dioxide incubator. On the 10th day after storage, the number of colonies of 5 drops per group at a dilution ratio that formed 10 to 100 colonies per drop under a stereomicroscope was measured and totaled to determine the number of colonies per 10 μL. The number of colonies in the other group at the same dilution ratio was determined in the same manner, and the average of the number of colonies in the second group was defined as the number of viable bacteria.

<Result>
Table 3 and FIG. 3 show the results of the viable count. FIG. 3 is a drawing comparing the detection sensitivity of the culture medium of the present invention, the animal raw material medium and the PPLO medium.

  The medium of the present invention was observed to have many colonies although no statistical difference was observed compared to the animal raw material medium and the PPLO medium, and it was confirmed that the detection sensitivity was excellent.

3. Proliferation of two other strains (KP-13 strain and S6 strain) of Mycoplasma galicepticam <Materials and methods>
The growth of the Mycoplasma gallisepticum KP-13 and S6 strains in the liquid medium of the present invention was confirmed.
A seed culture was inoculated into the liquid medium of the present invention in which each strain was adjusted to pH 7.3 ± 0.2, and left to stand at 37 ° C. for 24 hours to prepare an original culture solution. Each of the original cultures was continuously inoculated with 1/100 volume of the same medium.

  After 18 hours from the inoculation, the culture solution was collected every 6 hours and the viable cell count was measured.

  The double concentrated liquid medium of the present invention sterilized by filtration with a 0.2 μm filter and 2% by weight Bacto agar separately sterilized at 121 ± 2 ° C. were kept at 45-50 ° C. in a thermostatic bath, and then mixed in equal amounts. The mixed solution was poured into a plastic disposable sterilized Petri dish having a diameter of 60 mm and solidified, and then the lid was opened in a clean bench, turned over, and dried for 1 hour to obtain a viable count agar medium.

The collected culture solution was diluted 10-fold with the liquid medium of the present invention to 10 ^−5, and 10 μL of each dilution was divided into 5 drops and inoculated by dropping onto a viable count agar medium. Two groups were prepared for each step dilution, and after the inoculated liquid was absorbed into the agar, the Petri dish was turned over and stored in a 5% by volume carbon dioxide incubator. On the 7th day after storage, the number of colonies per group of 5 drops at a dilution rate of 10 to 100 colonies per drop under a stereomicroscope was measured and totaled to determine the number of colonies per 10 μL. The number of colonies in the other group at the same dilution ratio was determined in the same manner, and the average of the number of colonies in the second group was defined as the number of viable bacteria.

  In the culture medium of the present invention, it was confirmed that the mycoplasma gallicepticum KP-13 and S6 strains also grew.

It is drawing which compared the growth property in pH7.3 of this invention culture medium, animal raw material culture medium, and PPLO culture medium. It is drawing which compared the growth property in pH7.8 of this invention culture medium, animal raw material culture medium, and PPLO culture medium. It is drawing which compared the microbial count measurement culture medium detection sensitivity of this invention culture medium, animal raw material culture medium, and PPLO culture medium. It is drawing which showed the growth property of the mycoplasma gallisepticum KP-13 stock | strain and S6 strain | stump | stock in this invention culture medium.

Claims (2)

In purified water, purified soybean and / or defatted soybean treated with microorganism-derived enzyme 0.1 to 10% by weight, powdered yeast extract 0.1 to 10% by weight, sodium chloride 0.1 to 10% by weight, glucose 0.1 to 10% by weight and animal serum to 20 volume% in the presence of, features and to luma equalization plasma gallisepticum that was adjusted to be weakly acidic to weakly alkaline (pH 6.0 to 8.0) (Mycoplasma gallipticum) culture medium. Mycoplasma gallisepticum culture medium of claim 1 Symbol mounting, characterized in that solidified by adding agar.