BioVector® ATCC® 25978™ 欧洲亚硝化单胞菌模式菌株——产品技术说明书 (Product Datasheet)

1. 产品基本信息与类型鉴定 (Product & Type Strain Information)

• 菌株学名：Nitrosomonas europaea Winogradsky

• ATCC 编号：25978™

• 菌株核心编号：C-31 / Nm50 / DSM 28437

• 分类学地位：本物种乃至整个亚硝化单胞菌属（Nitrosomonas）的模式种（Type strain / Nomenclatural type）。所有新发现的氨氧化细菌进行分类学认定时，均需以此菌株作为金标准比对。

• 生物学来源：分离自陆地土壤（Soil）。

• 生理特征：专性化学自养菌，严格需氧，通过氧化环境中的氨产生亚硝酸盐获取能量。

• 产品形态：冷冻冰砖或冻干管（Frozen / Lyophilized format）。

2. 基因组测序与质控信息 (Genomic & Reference Profile)

• 测序状态：已完成高通量全基因组测序（Whole-genome sequenced strain）。

• 基因组大小：约 2.7 Mb。

• 测序质控标记：

  • NCBI RefSeq：GCF_900167395.1

  • 16S rRNA 序列登录号：AF353160 或 Z46927

3. 专属培养基及红线配方 (Medium Specification)

官方指定培养基：ATCC Medium #2265: Nitrosomonas Medium

• 物理环境：26.0 ℃，有氧/需氧环境（Aerobic）。

• 生化禁忌（红线）：自养菌对有机碳极其敏感，禁止加入任何浓度的葡萄糖、氨基酸或天然提取物（如酵母浸膏）。

• 玻璃器皿要求：微量重金属残留或洗涤剂碱性残留会对 C-31 株造成致死性毒性，所有培养瓶必须经过严格酸洗（如稀盐酸浸泡后超纯水冲洗）。

4. 传代操作与扩增流线 (Subculturing Protocol & Kinetics)

与常规细菌不同，ATCC 25978 具有双轨生长动力学特征（静置与震荡）：

• 传代周期（静置）：在静置（Static）状态下栽培时，菌体增殖较慢，通常需要 7 到 14 天 才能观察到丰富的生物量（Abundant growth）。

• 传代周期（加速震荡）：若放置于往复式或旋转式摇床上进行震荡培养，因大幅提升了溶氧效率，其倍增生长速率可显著缩短至 3 到 5 天。

• 推荐接种稀释比：采用 10% 比例大体积接种（即 10 mL 浓稠老菌液转接至 100 mL 新鲜 Medium 2265 中）。

• 菌种常规维持：培养至对数末期的成熟培养液，可直接放入 4 ℃ 冰箱 中进行避光短期封存，每 4 到 6 周必须全量传代一次。

5. 官方标准复苏双瓶操作法 (Standard ATCC Recovery - Two-Flask Technique)

由于模式菌株冻存后细胞壁较为脆弱，ATCC 官方推荐使用双瓶稀释法以确保复苏成功率：

1. 准备 两瓶 盛有 15 mL 灭菌、pH 稳态的 ATCC #2265 液体培养基的三角锥瓶（或透气细胞培养瓶），全程用铝箔纸包裹避光。

2. 从 -80 ℃ 或液氮中取出 ATCC 25978 冻存管，置于 37 ℃ 水浴中快速摇晃融化（60-90秒）。

3. 接种第一瓶（Flask #1）：在无菌条件下，将解冻后的全部菌悬液直接注入第一瓶 15 mL 培养基中。

4. 接种第二瓶（Flask #2，梯度稀释）：立即从第一瓶（Flask #1）中抽取 1.0 mL 混合液，转接至第二瓶 15 mL 新鲜培养基中。

   • 注：此步骤用于防止原冻存管中可能存在的局部底物抑制或保护剂残留浓度过高。

5. 孵育形态：将两瓶同时置于 26 ℃ 绝对黑暗 中孵育。如果使用的是透气细胞培养瓶（Tissue culture flask），必须将培养瓶平躺放置（Laid down），以最大化气-液接触面积，促进高效的空气交换。

6. 观察到液体由清亮转为轻微乳白浑浊（Turbidity increase）即表明复苏成功。

6. 生物安全与超低温冷冻保存 (Biosafety & Storage)

• 生物安全等级 (BSL)：BSL-1（最低风险级别）。对健康成年人类或动物不具备致病攻击性。

• 长期封存冷冻温度：必须存放在 -80 °C 或更低的超低温环境（推荐液氮气相）。

• 冷冻保护剂：可使用自养菌专用的无机盐保护剂或常规甘油混剂进行超低温冻存。

ATCC® 25978™ Nitrosomonas europaea Winogradsky C-31 (Type Strain) Product Datasheet

1. Product & Type Strain Information

• Scientific Name: Nitrosomonas europaea Winogradsky

• ATCC Number: 25978™

• Strain Designations: C-31 / Nm50 / DSM 28437

• Taxonomic Status: Nomenclatural Type strain of the species and genus. Serves as the ultimate taxonomic gold standard for ammonia-oxidizing bacteria (AOB) identification.

• Isolation Source: Isolated from terrestrial soil.

• Physiology: Obligate chemolithoautotroph, strictly aerobic; oxidizes ammonia to nitrite.

• Product Format: Frozen or Lyophilized vial.

2. Genomic Features

• Status: Whole-genome sequenced type strain.

• Genome Size: ~ 2.7 Mb.

• Reference Accessions:

  • NCBI RefSeq: GCF_900167395.1

  • 16S rRNA GenBank: AF353160 / Z46927

3. Growth Medium & Chemical Restrictions

• Designated Medium: ATCC Medium 2265: Nitrosomonas europaea medium.

• Incubation Parameters: 26 °C, Aerobic.

• Organic Restrictions: Autotrophic culture; do not add any organic carbon sources (e.g., glucose, peptone, or yeast extract) as they strongly inhibit growth.

• Glassware Warning: Highly sensitive to chemical or detergent residues; clean all flasks thoroughly with an acid wash followed by deionized water rinses.

4. Subculturing Protocol & Growth Kinetics

• Static Growth Kinetics: In static (unshaken) cultures, growth is slow, and abundant biomass or turbidity is typically evident in 7 to 14 days.

• Accelerated Shaking Kinetics: The growth rate can be significantly accelerated to 3 to 5 days by cultivating the vessels on a reciprocal or rotary shaker.

• Inoculum Volume: A 10% inoculum (e.g., 10 mL of dense culture into 100 mL of fresh medium) is strictly recommended.

• Maintenance Schedule: Transfer the culture every four to six weeks, and store the fully grown stock culture at 4 °C in the absolute dark.

5. Official ATCC Two-Flask Recovery Technique

To bypass substrate inhibition or local cryoprotectant toxicity during thawing, utilize the following mandatory two-flask dilution procedure:

1. Prepare two separate Erlenmeyer flasks or tissue culture flasks containing 15 mL of sterile ATCC broth #2265. Ensure they are completely wrapped in aluminum foil to block out light.

2. Rapidly thaw the frozen vial by swirling it in a 37 °C water bath for 60 to 90 seconds.

3. Inoculate Flask #1: Aseptically transfer the entire contents of the thawed vial into the first flask containing 15 mL of medium.

4. Inoculate Flask #2 (Serial Dilution): Immediately pipet 1.0 mL from Flask #1 and transfer it into the second flask containing 15 mL of fresh medium.

5. Incubation Setup: Incubate both flasks in the dark at 26 °C. If using tissue culture flasks, the flasks must be laid down horizontally during incubation to optimize gas-liquid surface interaction and promote proper air exchange.

6. Cells are multiplying successfully once an increase in milk-like turbidity is visible under lighting conditions.

6. Biosafety & Storage Standards

• Biosafety Level: BSL-1. Non-pathogenic to healthy adult humans or animals.

• Long-Term Preservation: Cryovials must be stored in a freezer maintained at -80 °C or colder (or vapor-phase liquid nitrogen).Cells may be preserved by freezing with a validated autotrophic cryoprotectant.

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