JP4210674B2 - Mycoplasma gallicepticum culture medium - Google Patents
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- 239000001963 growth medium Substances 0.000 title claims description 23
- 241000204031 Mycoplasma Species 0.000 title claims description 13
- 241001465754 Metazoa Species 0.000 claims description 23
- 229920001817 Agar Polymers 0.000 claims description 17
- 239000008272 agar Substances 0.000 claims description 17
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 244000068988 Glycine max Species 0.000 claims description 5
- 235000010469 Glycine max Nutrition 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- 241000204022 Mycoplasma gallisepticum Species 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 239000008213 purified water Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 241000023320 Luma <angiosperm> Species 0.000 claims 1
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 claims 1
- 239000002609 medium Substances 0.000 description 51
- 239000007788 liquid Substances 0.000 description 15
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- 108090000623 proteins and genes Proteins 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 239000002994 raw material Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 244000052769 pathogen Species 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 241000287828 Gallus gallus Species 0.000 description 4
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- 238000001514 detection method Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 235000013372 meat Nutrition 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 241000271566 Aves Species 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
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- 229960005486 vaccine Drugs 0.000 description 3
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- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 240000004713 Pisum sativum Species 0.000 description 1
- 235000010582 Pisum sativum Nutrition 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 240000006677 Vicia faba Species 0.000 description 1
- 235000010749 Vicia faba Nutrition 0.000 description 1
- 235000002098 Vicia faba var. major Nutrition 0.000 description 1
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- 150000001720 carbohydrates Chemical class 0.000 description 1
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- 210000000416 exudates and transudate Anatomy 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、タンパク源が植物由来タンパク質であるマイコプラズマ・ガリセプティカム(Mycoplasma gallisepticum)培養用培地に関する。当該培地は、マイコプラズマ・ガリセプティカムの純粋培養及び生菌数計測(集落数計測)に有用である。 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).
家禽等の鳥類に感染するマイコプラズマ・ガリセプティカムを培養するための培地には、従来、タンパク源として獣肉エキス、鶏肉ブイヨン、牛乳由来カゼイン等の動物由来原料を含むペプトン水に、マイコプラズマ・ガリセプティカムの成育に必須の血清成分を含めた添加物として、動物血清、酵母エキス、塩類及び糖類を加えた液体培地や寒天固化培地が用いられてきた(例えば、非特許文献1参照。)。マイコプラズマ・ガリセプティカムを培養する培地にタンパク源として添加されてきた、これらの獣肉エキス、鶏肉ブイヨン、牛乳由来カゼイン等は、動物の生産物や臓器から浸出あるいは抽出した物質である。培養したマイコプラズマ・ガリセプティカムは、それが家禽等の鳥類に感染して当該鳥類が発病するのを防止及び軽減するためのワクチンを製造するためにも使用されるほか、ヒトや動物の診断薬等にも使用される。
ところが近年、前記のタンパク源としての原料が由来する動物特に反芻動物から人に伝播性がある牛海綿状悩症(BSE)病原体が発見された。そして、BSE病原体を含む動物原料を培地として培養されたマイコプラズマ・ガリセプティカムから製造されたワクチンには、BSE病原体混入のおそれがあって、それが家禽等にワクチンとして接種されると、肉及び卵などの生産物を介してヒトが摂食して病原体に曝露される危険性がある。さらに、動物由来原料には、一般的にみて、病原微生物の伝播や物質汚染の危険性が大きい。 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.
そこで、本発明の目的は、マイコプラズマ・ガリセプティカム培養用培地のタンパク源として、動物由来原料を使用しないことで、動物由来原料使用に起因する病原微生物の伝播や物質汚染やBSE病原体混入の危険を回避し、以って、肉及び卵などの生産物を介してヒトが摂食して病原体に曝露される危険性をなくすることである。 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.
本発明者等は、マイコプラズマ・ガリセプティカム培養用培地にタンパク源として存在させるタンパク質を、従来の動物原料由来タンパク質から植物原料由来タンパク質に代替しても、従来のものと同等以上にマイコプラズマ・ガリセプティカムが良好に増殖することを見出し、本発明を完成するに至った。すなわち、本発明は、タンパク源が植物由来タンパク質であることを特徴とするマイコプラズマ・ガリセプティカム培養用培地に関するものである。 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.
本発明において使用される植物原料由来タンパク質の原料としては種々のものがあるが、大豆、そら豆、えんどう豆等の豆類が特に好ましい。 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.
以下、本発明の具体的な実施形態を説明する。 Hereinafter, specific embodiments of the present invention will be described.
精製水中に、非加熱ニワトリ血清が1〜20容積%、大豆精製物を微生物由来酵素で分解した「ポリペプトンN」(日本製薬社製)及び/又は脱脂大豆を微生物由来酵素で分解した「ポリペプトンNS」(日本製薬社製)が合計で0.1〜10重量%、粉末酵母エキスが0.1〜10重量%、塩化ナトリウムが0.1〜10重量%、ブドウ糖が0.1〜10重量%となるように加え、それに水酸化ナトリウムを加えて弱酸性から弱アルカリ性(pH6.0〜8.0)となるように調整し、ろ過あるいは高圧蒸気滅菌した液体、ないしは、これに寒天を加えて固形化したものを、マイコプラズマ・ガリセプティカム培養用培地とする。本発明において、重量%はw/v%を表すものとする。 "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.液体培地における増殖性の比較
<材料と方法>
本発明培地、本発明培地の「ポリペプトンN」に替えて牛乳カゼイン由来タンパクを酵素分解した「ポリペプトン」(日本製薬社製)を同濃度で加えた培地(以下「動物原料培地」ということがある。)及び20容積%ウマ血清加変法PPLO培地(ウシ心臓浸出液を含む栄研化学社製ハートインフュージョンブイヨン25.0g/1,000mL、粉末酵母エキス0.5重量%、ブドウ糖0.5重量%、以下「PPLO培地」ということがある。)に、それぞれ、10重量%水酸化ナトリウムを加えて、pH7.3±0.1及びpH7.8±0.1に調整したものを被検培地とした。なお、「変法」の語句を付加したのは、本発明培地と比較するために、通常のウマ血清加PPLO培地ではブドウ糖0.1重量%とされるところ、ブドウ糖0.5重量%としたためである。
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.
マイコプラズマ・ガリセプティカム1RF株種菌をpH7.3±0.1に調整した本発明培地に接種した。 Mycoplasma gallisepticum 1RF inoculum was inoculated into the medium of the present invention adjusted to pH 7.3 ± 0.1.
37℃で24時間静置培養して元培養菌液を作成した。元培養菌液をそれぞれの被検培地に1/100量を接種した。 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.
接種後16時間目と24時間目、以後12時間目ごとに培養液を採取し、生菌数を測定した。 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.
具体的な操作は下記のとおりである。
0.2μmのフィルターでろ過滅菌した本発明培地の2倍濃厚液体培地を調製した。この培地と別途121±2℃で滅菌した2重量%バクト寒天(Bacto Agar)とをそれぞれ恒温槽において45〜50℃に保温した後等量混合し、混合液を直径60mmのプラスチック製ディスポ滅菌ペトリ皿に5mLを注いで固化した。固化後クリーンベンチ内に蓋を開いて転倒して1時間置いて乾燥し、生菌数計測寒天培地とした。
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.
採取した培養液を本発明液体培地で10−5まで10倍階段希釈し、各希釈10μLを5滴に分けて生菌数計測寒天培地上に滴下して接種した。階段希釈ごとにこれを2群作成し、接種した液が寒天に吸収された後、ペトリ皿を転倒して5容積%炭酸ガスインキュベータに収納した。収納後10日目に実体顕微鏡下で1滴当り10〜100個の集落を形成した希釈倍率の1群5滴の集落数を計測して合計し、10μL当りの集落数を求めた。同希釈倍率のもう1群の集落数を同様に求め、2群の集落数の平均を生菌数とした。 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.
<結果>
表1、表2、図1及び図2に生菌数の成績を示した。表1及び図1は、本発明培地、動物原料培地及びPPLO培地のpH7.3における増殖性を比較した表及び図面であり、表2及び図2は、本発明培地、動物原料培地及びPPLO培地のpH7.8における増殖性を比較した表及び図面である。
<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.
pH7.3の本発明培地で培養するとき、接種後36時間目に最大生菌数4.5×108 cfu/mLとなった。動物原料培地では60時間目に1.9×108 cfu/mL、PPLO培地では72時間目に1.1×109 cfu/mLであった。 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.
pH7.8の本発明培地で培養するとき、接種後48時間目に最大生菌数9.3×108 cfu/mLとなった。動物原料培地では60時間目に3.1×108 cfu/mL、PPLO培地では96時間目に2.1×109 cfu/mLであった。 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.
本発明培地はいずれのpHでも、動物原料及びPPLO培地と比べ速やかな増殖が認められた。最大生菌数はPPLO培地には及ばないものの良好に増殖することが認められた。 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.寒天培地における検出感度の比較
<材料と方法>
寒天培地を得るために、まず、0.2μmのフィルターでろ過滅菌した本発明培地、動物原料培地及びPPLO培地それぞれの2倍濃厚液体培地を調製した。この培地と別途121±2℃で滅菌した2重量%Bacto Agarとをそれぞれ恒温槽において45〜50℃に保温した後等量混合した。混合液を直径60mmのプラスチック製ディスポ滅菌ペトリ皿に5mLを注いで固化した後、クリーンベンチ内に蓋を開いて転倒して1時間置いて乾燥し、生菌数計測寒天培地とした。
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.
マイコプラズマ・ガリセプティカム1RF株種菌をpH7.3±0.2に調整した本発明培地に接種し、37℃で24時間静置培養して元培養菌液を作成した。元培養菌液を本発明培地に1/100量を接種し、16時間目、以後4時間目ごとに28時間目まで、次いで36時間目、以後6時間目ごとに48時間目まで採取した培養液を試料とした。 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.
試料を本発明液体培地で10−5まで10倍階段希釈し、各希釈10μLを5滴に分けて各寒天培地上に滴下して接種した。階段希釈ごとにこれを2群作成し、接種した液が寒天に吸収された後、ペトリ皿を転倒して5容積%炭酸ガスインキュベータに収納した。収納後10日目に実体顕微鏡下で1滴当り10〜100個の集落を形成した希釈倍率の1群5滴の集落数を計測して合計し、10μL当りの集落数を求めた。同希釈倍率のもう1群の集落数を同様に求め、2群の集落数の平均を生菌数とした。 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.
<結果>
表3及び図3に生菌数の成績を示した。図3は、本発明培地、動物原料培地及びPPLO培地の菌数測定培地検出感度を比較した図面である。
<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.
本発明培地は動物原料培地及びPPLO培地と比べて、統計学的な差は認められないものの集落が多く観察され、検出感度が優れていることを確認した。 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.マイコプラズマ・ガリセプティカムの他の2株(KP−13株及びS6株)の増殖性
<材料と方法>
マイコプラズマ・ガリセプティカムKP−13及びS6株について本発明液体培地における増殖性を確認した。
各株をpH7.3±0.2に調整した本発明の液体培地に種菌を接種し、37℃で24時間静置培養して元培養菌液を作成した。それぞれ元培養した菌液を連続して同培地に1/100量を接種した。
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.
接種後18時間目以降6時間ごとに培養液を採取して生菌数を測定した。 After 18 hours from the inoculation, the culture solution was collected every 6 hours and the viable cell count was measured.
0.2μmのフィルターでろ過滅菌した本発明2倍濃厚液体培地及び別途121±2℃で滅菌した2重量%バクト寒天をそれぞれ恒温槽において45〜50℃に保温した後等量混合した。混合液を直径60mmのプラスチック製ディスポ滅菌ペトリ皿に5mLを注いで固化した後、クリーンベンチ内に蓋を開いて転倒して1時間置いて乾燥し、生菌数計測寒天培地とした。 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.
採取した培養液を本発明液体培地で10−5まで10倍階段希釈し、各希釈10μLを5滴に分けて生菌数計測寒天培地上に滴下して接種した。階段希釈ごとにこれを2群作成し、接種した液が寒天に吸収された後、ペトリ皿を転倒して5容積%炭酸ガスインキュベータに収納した。収納後7日目に実体顕微鏡下で1滴当り10〜100個の集落を形成した希釈倍率の1群5滴の集落数を計測して合計し、10μL当りの集落数を求めた。同希釈倍率のもう1群の集落数を同様に求め、2群の集落数の平均を生菌数とした。 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.
本発明培地において、マイコプラズマ・ガリセプティカムKP−13株及びS6株においても増殖することを確認した。 In the culture medium of the present invention, it was confirmed that the mycoplasma gallicepticum KP-13 and S6 strains also grew.
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