BioVector® 副溶血性弧菌 RIMD 2210633 标准菌株

BioVector® 副溶血性弧菌 RIMD 2210633 标准菌株——产品技术说明书

1 菌株基本信息与重大科研背景产品名称：BioVector® 副溶血性弧菌 RIMD 2210633 标准菌株常用别名：Vibrio parahaemolyticus RIMD 2210633，RIMD2210633，副溶血弧菌大坂株生物学分类：细菌界 Bacteria / 变形菌门 Proteobacteria / 弧菌科 Vibrionaceae / 弧菌属 Vibrio分离来源：1996年从日本大坂急性肠胃炎患者样本中分离出的临床大流行爆发株（血清型 O3:K6）形态特征：革兰氏阴性（G-）多形性弧菌，呈短杆状或单弯曲逗点状，极端单鞭毛，运动能力极强。核心科研与国际里程碑价值：该菌株是全球首个完成全基因组测序的副溶血性弧菌标杆参考株（由日本研究团队于2003年完成测序）。基因组包含两条独特的环状染色体。它是国际上研究大流行 O3:K6 血清型弧菌毒力进化、三型分泌系统（TSS3-1 和 T6SS 等分泌系统）、环境适应机制、基因组岛（Genomic Islands）以及全球食品安全分子流行病学溯源的公认第一标准底盘。

2 核心生理生化特性与三大致病系统RIMD 2210633 菌株作为测序标杆，其体内蕴含着极其清晰且复杂的致病机制：

严格专性嗜盐性：必须在含有氯化钠 NaCl 的环境平衡下生存，最适 NaCl 生长浓度为 3%，完全无盐环境下会迅速发生溶菌死亡。两大核心毒力基因：该菌株在基因组上同时携带 tdh1 基因和 tdh2 基因，能够大量高效地分泌耐热溶血素（Thermostable Direct Hemolysin, TDH）。在神奈川现象（Kanagawa Phenomenon）测试中呈现强阳性（即在 Wagatsuma 琼脂平板上使人红细胞发生彻底的 beta-溶血）。双三型分泌系统（T3SS1 / T3SS2）：T3SS1（位于染色体 I）：主要负责介导对宿主上皮细胞的细胞毒性，引发细胞自噬或凋亡。T3SS2（位于染色体 II）：主要介导肠道毒性，是导致临床剧烈腹泻、肠粘膜损伤及系统性肠炎的核心驱动力。

3 专用高盐培养基配方规范

红线警告：培养基配方必须人为额外补加 3% 的 NaCl，常规普通 LB 或普通营养肉汤无法维持该菌株存活。

A 3% NaCl 营养琼脂平板/肉汤配方（用于日常日常传代与常规扩增）基础成分：牛肉膏 3.0 g，蛋白胨 10.0 g。核心专性添加剂：纯氯化钠 NaCl 30.0 g（即 3% 终浓度）。琼脂添加（仅限平板固化）：琼脂粉 15.0 g。溶剂：蒸馏水或去离子水 1000 mL。pH 调节：使用高浓度 NaOH 调节 pH 值至 8.0 到 8.5（该菌嗜碱怕酸）。灭菌规范：121 ℃ 高压蒸汽灭菌 15 分钟。

B TCBS 琼脂培养基配方（专性选择性分离/鉴定平板）全称：硫代硫酸盐柠檬酸盐胆盐蔗糖琼脂培养基。生长表型：由于 RIMD 2210633 不具备发酵蔗糖的能力，在此平板上培养 24 小时后，会形成直径为 2 至 5 毫米、圆形、表面湿润、中心折光良好的蓝绿色或深绿色菌落（借此可与霍乱弧菌等发酵蔗糖呈黄色的菌落进行明确区分）。

C 菌株长期冻存保护液标准配方：含 3% NaCl 的营养肉汤 80% + 细胞/细菌级纯甘油 20%。混匀后高压灭菌，与对数生长期的菌液等体积混合后深低温保存。

4 物理环境控制参数培养温度：37.0 ℃（最适生长温度范围 36.0 ℃ 至 38.0 ℃）气相环境：常规常压无菌空气，需氧生长pH 范围：8.0 - 8.5（最适碱性范围）倍增速度：在营养充足、3% 盐度且 pH 适宜的液体肉汤中，该菌分裂极快，代时可缩短至十几分钟，可在 6-8 小时内使清亮的肉汤变为极度浑浊。

5 菌种活化、传代操作规范传代临界点：固体平板接种后培养 18-24 小时，当单个独立菌落生长完全时，即可用于后续实验或转种。切勿在 37 ℃ 下连续放置超过 48 小时，否则菌落会迅速进入衰亡期并发生自溶。

标准平板划线传代步骤：1 从超低温冰箱或冷冻干燥管中获取菌种源。2 在超净台内，点燃酒精灯。使用金属接种环在火焰上烧灼至通红，随后在平板无菌区域边缘贴靠冷却。3 蘸取少许菌液或挑取单个独立菌落，在 3% NaCl 营养琼脂平板或 TCBS 平板上执行标准三段或四段法划线，以获得稀释的单菌落。4 划线完毕后，盖上皿盖，将平板倒置（皿底朝上），放入 37 ℃ 恒温培养箱中暗培养 18 到 24 小时。

6 冷冻干燥菌种管（安瓿管）的开启与复苏恢复流线1 准备 1 支盛有 4.0 到 5.0 mL 预热的 3% NaCl 营养肉汤管。2 拿起副溶血性弧菌 RIMD 2210633 冻干安瓿管，用 75% 酒精棉球擦拭其外壁进行表面消毒。3 使用砂轮在安瓿管顶端（有棉签一端）轻轻划一圈细痕，随后用一滴无菌水或微热的玻璃棒触碰划痕处，使管壁产生微小裂纹。4 用无菌镊子轻轻敲碎管帽，或用垫有无菌纱布的手指将管帽折断。5 用无菌移液管吸取 0.5 mL 预热的 3% NaCl 营养肉汤，缓缓注入安瓿管底部，轻柔吹吸数次，使干燥的菌体粉末完全溶解并呈均匀浑浊状。6 吸出全部菌悬液，回注到剩余的 3% NaCl 营养肉汤管中，充分混匀。7 从中吸取 100 uL 菌液，接种到 3% NaCl 营养琼脂平板或 TCBS 平板上进行涂布/划线，以确保获取纯净的单菌落。8 将液体肉汤管与固体平板一并放入 37 ℃ 培养箱中培养 18-24 小时。

7 长期封存、生物安全与质控规范生物安全级别：BSL-2（二级生物安全柜操作）。RIMD 2210633 为典型的高毒力临床大流行株，日常实验必须在具备二级生物安全防护屏障的实验室内进行。所有接触过该菌的培养基、枪头、移液管及平板必须经过 121 ℃ 高压灭菌至少 30 分钟后方可废弃，严防泄露污染环境。超低温长期保存：长期保存必须置于 -80 ℃ 超低温冰箱（可维持 1-2 年）或永久存放于液氮罐（-196 ℃）中。严禁在 4 ℃ 普通冰箱中长期保存液体菌种，该菌在 4 ℃ 环境下极易发生“可活不可培养状态”（VBNC）或直接大批自溶死亡。基因组质控复核（可选）：由于该菌株是全基因组测序的金标准，为了验证实验过程中未发生菌株混淆，可以定期提取该菌基因组 DNA，通过 PCR 特异性扩增其 T3SS1（如 vscC 基因）和 T3SS2（如 vscC2 基因）的标记性保守序列，双重阳性且嗜盐平板生长特征典型，方可确证其 RIMD 2210633 的本源血统。

BioVector® Vibrio parahaemolyticus RIMD 2210633 Standard Genomic Reference Strain Product Datasheet

1 Strain and Identification General InformationProduct Name: BioVector® Vibrio parahaemolyticus RIMD 2210633 Standard Reference StrainSynonyms: Vibrio parahaemolyticus RIMD 2210633, RIMD2210633, Osaka Clinical Pandemical StrainBiological Taxonomy: Kingdom Bacteria / Phylum Proteobacteria / Family Vibrionaceae / Genus VibrioIsolation Source: Isolated from a patient with acute gastroenteritis during the clinical pandemic outbreak in Osaka, Japan, 1996 (Serotype O3:K6)Morphological Characteristics: Gram-negative (G-), pleomorphic curved rods, short bacilli or comma-shaped architecture. Possesses a single polar flagellum driving energetic swarming and swimming motility.Core Research & International Milestone Significance: This item stands as the world first fully sequenced genome reference strain for Vibrio parahaemolyticus (sequenced by a Japanese research consortium in 2003). Its genome features two distinct circular chromosomes. It serves as the primary international benchmark model for studying the evolution of pandemic O3:K6 serotypes, Type III Secretion Systems (T3SS-1 and T3SS-2 pathways), environmental fitness drivers, genomic pathogenicity islands, and global food safety molecular epidemiological tracing.

2 Core Physiological-Biochemical Profiling and Pathogenic MachineriesVibrio parahaemolyticus RIMD 2210633 manifests classic halophilic dependencies alongside a fully characterized virulence gene repertoire:

Obligate Halophilic Kinetics: Demands sodium chloride (NaCl) osmotic balances for metabolic preservation, showing a growth optimum at 3% NaCl concentration. Complete absence of salinity in the media triggers immediate wall destabilization and rapid autolytic lysis.Dual Core Virulence Transcripts: Its genome co-harbors both tdh1 and tdh2 loci, leading to heavy production and secretion of Thermostable Direct Hemolysin (TDH). It scores a potent positive mark in the Kanagawa Phenomenon (KP) assay, inducing clear beta-hemolytic rings on Wagatsuma agar matrices packed with human erythrocytes.Dual Type III Secretion Apparatuses (T3SS1 / T3SS2):T3SS1 (Localized on Chromosome I): Predominantly drives broad cytotoxicity against host epithelial structures, inducing autophagy or apoptotic cascades.T3SS2 (Localized on Chromosome II): Functions as the primary driver for enterotoxicity, directly causing fluid accumulation, mucosal structural sloughing, and the clinical symptoms of acute diarrheal enteritis.

3 Dedicated Halophilic Media Formulations

CRITICAL WARNING: All custom or standard media recipes must be manually updated to contain a 3% final concentration of NaCl. Utilizing traditional Nutrient Broth or LB formulas without updating salt metrics will alter osmotic balances, causing immediate cell wall lysis and complete culture death.

A 3% NaCl Nutrient Agar / Broth Base (For Routine Expansion and Stock Maintenance)Basal Macro-Nutrients: Beef Extract 3.0 g, Peptone 10.0 g.Halophilic Core Additive: Sodium Chloride (NaCl) 30.0 g (yielding a 3% final concentration).Solidifying Agar Matrix (For plates only): Agar powder 15.0 g.Solvent Base: Distilled or Deionized Water 1000 mL.pH Standardization: Calibrate utilizing NaOH to hit a final alkaline index of 8.0 to 8.5 (The strain is highly alkaliphilic; acidic shifts cause rapid autolysis).Sterilization Metric: Autoclave at 121 ℃ for 15 minutes.

B TCBS Agar Formulation (Selective Isolation and Diagnostic Plates)Full Nomenclature: Thiosulfate Citrate Bile Salts Sucrose Agar.Selective Diagnostic Output: Because RIMD 2210633 lacks sucrose-fermenting machinery, it produces distinct 2 to 5 mm round, smooth, blue-green or deep green colonies after 24 hours of incubation (distinguishing it from Vibrio cholerae, which produces yellow colonies via sucrose utilization).

C Standard Long-Term Cryopreservation FluidGlycerol Freezing Blend: 80% Nutrient Broth supplemented with 3% NaCl + 20% Analytical/Bacterial Grade Pure Glycerol. Mix thoroughly and autoclave prior to blending with log-phase cultures for ultra-low temperature banking.

4 Controlled Culture Environmental ParametersIncubation Temperature: 37.0 ℃ (Optimal operating window spans 36.0 ℃ to 38.0 ℃)Gas Phase Composition: Standard aerobic atmospheric air environmentpH Workspace Parameters: 8.0 to 8.5 (Strictly alkaline preference)Replication Velocity: Under optimized 3% salinity and alkaline baselines, this organism splits rapidly; generation times can drop to under 20 minutes, converting a crystal-clear broth into a turbid dense state within 6 to 8 hours.

5 Subculturing Passaging Protocols and TimelinesPassaging Threshold: Harvest or transfer colonies from solid plates precisely at 18-24 hours post-inoculation when single colonies are fully realized. Do not store plates at 37 ℃ past 48 hours, as cultures slide rapidly into death phases and undergo active autolysis.

Standard Plate Quadrant Streak Method:1 Retrieve the active master stock or lyophilizate vial from cold storage.2 Within a certified biosafety workstation, ignite the bunsen burner. Heat the metal inoculating loop until glowing red, then cool it completely by touching a sterile border area of the agar matrix.3 Dip into the liquid starter suspension or touch a single isolated colony, and perform standard three- or four-quadrant streaking across a fresh 3% NaCl Nutrient Agar or TCBS plate to isolate single entries.4 Close the plate cover, invert the plate upside down (agar side facing upward), and place it inside the 37 ℃ incubator for 18 to 24 hours.

6 Rehydration and Recovery of Lyophilized Freeze-Dried Vials (Ampoules)1 Pre-warm a sterile tube containing 4.0 to 5.0 mL of 3% NaCl Nutrient Broth.2 Take the lyophilized Vibrio parahaemolyticus RIMD 2210633 glass ampoule and disinfect its exterior surface utilizing a 75% ethanol wipe.3 Use a sterile ampoule file or diamond-tipped cutter to score a light groove around the neck near the cotton plug end. Touch a drop of sterile water or a heated glass rod to the scored line to induce a controlled microscopic fracture.4 Use sterile forceps to gently break away the glass cap tip, or wrap with a sterile gauze pad to snap the neck open safely.5 Utilize a sterile pipette to transfer 0.5 mL of the pre-warmed 3% NaCl Nutrient Broth directly into the bottom of the open ampoule. Pipette up and down gently until the pellet powder dissolves completely into a uniform suspension.6 Draw up the entire suspension fluid and return it to the remaining 3% NaCl Nutrient Broth tube; mix thoroughly.7 Extract a 100 uL aliquot of the liquid suspension and spread it onto a 3% NaCl Nutrient Agar or TCBS plate to verify purity and capture distinct colony picks.8 Incubate both the liquid broth tube and solid plates concurrently at 37 ℃ for 18-24 hours.

7 Long-Term Storage, Biosafety and Quality ControlsBiosafety Level: BSL-2 (Class II Biosafety Cabinet Operations). RIMD 2210633 is a fully documented high-virulence clinical pandemic isolate; all laboratory manipulation must be constrained within an approved Level 2 containment facility. All spent cultures, plastics, pipettes, and plates must undergo a full 30-minute verification cycle at 121 ℃ autoclaving before entering biohazard disposal streams.Long-Term Cryo-Banking: Must be preserved inside a -80 ℃ ultra-low freezer (viable for 1-2 years) or stored indefinitely within liquid nitrogen containers (-196 ℃). Do not maintain liquid plates or tubes inside standard 4 ℃ refrigerators for prolonged periods; low temperatures induce a Viable But Non-Culturable (VBNC) state or cause complete cellular death.Genomic Quality Control Identity Assay (Optional): As a global master sequence strain, cross-contamination or identity drifts can be monitored via targeted PCR assays. Periodically isolate genomic DNA and confirm the dual presence of T3SS1 machinery (e.g., vscC gene amplification) and T3SS2 architecture (e.g., vscC2 gene amplification). Conclusive identity is satisfied when both targets generate strong positive bands coupled with standard Gram-negative halophilic profiles.

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