Vibrio parahaemolyticus副溶血性弧菌 20130629002S01 BioVector® 标准菌株

1 菌株基本信息与生物学背景产品名称：BioVector® 副溶血性弧菌 20130629002S01 标准菌株常用别名：Vibrio parahaemolyticus 20130629002S01，副溶血弧菌 002S01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：海洋生物、受污染海产品或临床腹泻患者标本形态特征：革兰氏阴性菌（G-），显微镜下呈多形性，主要为短杆状、弧状或逗点状，无芽孢，具有极生单鞭毛，运动极其活泼。核心科研与工业价值：该菌株是一株具有特定分子分型背景的副溶血性弧菌标准株。副溶血性弧菌是导致海产品源性感染、食物中毒以及急性胃肠炎的国际首要致病菌。该菌株广泛应用于食品安全致病菌定量质控、药敏检测试验（AST）、新型致病机制研究、海水养殖鱼虾类病原学鉴定以及食品防腐剂效能评价。

2 核心生理生化特性与致病因子副溶血性弧菌 20130629002S01 表现出极其典型的嗜盐性以及特定的致病特征：

核心嗜盐特性：属于高度专性嗜盐性细菌。在完全不含氯化钠（NaCl）的普通培养基中绝对无法生长。其生长所需的 NaCl 浓度范围在 1% 至 8% 之间，最适生长氯化钠浓度为 3%。致病因子鉴定表型：副溶血性弧菌的核心毒力因子包括耐热溶血素（TDH）和/或 tdh 相关溶血素（TRH）。该菌株在日常质控中通常通过神奈川现象（Kanagawa Phenomenon，KP）检测试验（即在含高盐和人红细胞的 Wagatsuma 琼脂平板上观察是否产生特异性 beta-溶血环）或 PCR 扩增 tdh/trh 基因来锁定制定的毒力谱系。常规生化鉴定表型：氧化酶试验阳性；吲哚试验阳性；不发酵蔗糖，但发酵葡萄糖（不产气）。在含有 3% NaCl 的三糖铁（TSI）培养基中表现为底层变黄（产酸）、斜面不变色（不发酵乳糖/蔗糖）、不产硫化氢。

3 专用高盐培养基配方规范

红线警告：配置培养基时切勿漏加或少加氯化钠。如果使用标准的普通营养肉汤（Nutrient Broth）或 LB 培养基，必须人为额外额外补加 3% 的 NaCl，否则该菌株将由于渗透压失衡导致菌体裂解死亡。

A 3% NaCl 营养琼脂平板/肉汤配方（用于日常扩增、转种与维持）基础成分：牛肉膏 3.0 g，蛋白胨 10.0 g。关键嗜盐添加剂：氯化钠 NaCl 30.0 g（即 3% 终浓度）。琼脂补剂（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：用 NaOH 调节 pH 值至 8.0 到 8.5（该菌高度嗜碱，在酸性环境中极易死亡）。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（Thiosulfate Citrate Bile Salts Sucrose Agar）。培养基特征：由于副溶血性弧菌不发酵蔗糖，在该平板上生长 24 小时后，会形成直径为 2 至 4 毫米、圆形、边缘整齐、中心折光良好的蓝绿色或绿色菌落（而发酵蔗糖的霍乱弧菌则呈现黄色菌落）。

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%。充分混匀后高压灭菌，用于菌种深低温保存。

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 20130629002S01 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）。副溶血性弧菌属于国家规定的二类或三类致病菌（视具体菌株毒力和感染途径而定），日常操作必须在二级生物安全实验室内进行。所有接触过该菌的枪头、培养基、平板必须经过 121 ℃ 高压灭菌 30 分钟后方可作为医疗废物处理，严防污染外环境。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。嗜盐性与纯度复核：每隔 5-10 代，应将菌株同时接种至“不含 NaCl 的普通琼脂平板”和“含 3% NaCl 的琼脂平板”上。若不含盐平板完全不生长，而高盐平板生长出特征性边缘整齐、灰白色、湿润的单菌落，且革兰氏染色显示为阴性弧菌，方可确证该菌株未受到杂菌污染且嗜盐表型完好。

BioVector® Vibrio parahaemolyticus 20130629002S01 Standard Strain Product Datasheet

1 Strain and Identification General InformationProduct Name: BioVector® Vibrio parahaemolyticus 20130629002S01 Standard StrainSynonyms: Vibrio parahaemolyticus 20130629002S01, V. parahaemolyticus 002S01Biological Taxonomy: Kingdom Bacteria / Phylum Proteobacteria / Family Vibrionaceae / Genus VibrioIsolation Source: Marine environments, contaminated seafood matrix, or clinical gastroenteritis stool samplesMorphological Characteristics: Gram-negative (G-), pleomorphic short rods, curved or comma-shaped bacilli. Asporogenous (non-spore-forming), possesses a single polar flagellum exhibiting highly active motility.Core Research & Industrial Significance: This specific item represents a standard reference strain of Vibrio parahaemolyticus with a documented molecular profiling background. As a primary global pathogen responsible for seafood-borne gastroenteritis and acute food poisoning outbreaks, this strain is utilized heavily for food safety quantitative quality controls, antimicrobial susceptibility testing (AST), virulence mechanism mapping, marine aquaculture disease tracking (shrimp/fish pathology), and evaluation of food preservative efficacy.

2 Core Physiological-Biochemical Profiling and Virulence FactorsVibrio parahaemolyticus 20130629002S01 exhibits strict halophilic properties and distinct pathogenic profiles:

Strict Halophilic Dependency: Functions as an obligate halophilic bacterium. It cannot survive or replicate in media missing sodium chloride (NaCl). The operational salinity range spans 1% to 8% NaCl, with an absolute growth optimum at 3% NaCl density.Virulence Matrix Marker: Major pathogenicity is dictated by the expression of Thermostable Direct Hemolysin (TDH) and/or TDH-Related Hemolysin (TRH). This strain's virulence profile is verified routinely via the Kanagawa Phenomenon (KP) assay (beta-hemolysis zone visualization on Wagatsuma agar rich in human erythrocytes and high salt) or through direct PCR targeted amplification of tdh and trh gene sequences.Standard Biochemical Reaction Benchmarks: Oxidase positive; Indole positive; Sucrose non-fermenting, but Glucose fermenting (without gas evolution). On a Triple Sugar Iron (TSI) matrix fortified with 3% NaCl, it yields an acid butt (yellow), an alkaline slant (red/unchanged due to lack of lactose/sucrose fermentation), and zero Hydrogen Sulfide (H2S) production.

3 Dedicated Halophilic Media Formulations

CRITICAL WARNING: Always ensure that sodium chloride is incorporated into all custom medium recipes. Utilizing standard 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 Vibrio parahaemolyticus lacks sucrose-fermenting machinery, it produces distinct 2 to 4 mm round, smooth, blue-green or 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 20130629002S01 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). This strain must be manipulated within an approved Level 2 containment suite due to its classification as a human foodborne pathogen. All spent cultures, plastics, pipettes, and plates must undergo a full 30-minute validation 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.Halophilic and Purity Validation QC: Every 5-10 passages, verify identity by plating samples simultaneously on a "0% NaCl standard nutrient agar plate" and a "3% NaCl standard nutrient agar plate." Conclusive quality control is satisfied when the 0% NaCl matrix remains completely sterile, while the 3% NaCl matrix demonstrates characteristic translucent, off-white, circular Gram-negative vibrio colonies.