1 菌株基本信息與生物學(xué)背景產(chǎn)品名稱：BioVector? 副溶血性弧菌 20130629002S01 標(biāo)準(zhǔn)菌株常用別名：Vibrio parahaemolyticus 20130629002S01，副溶血弧菌 002S01生物學(xué)分類：細(xì)菌界 Bacteria / 變形菌門 Proteobacteria / 弧菌科 Vibrionaceae / 弧菌屬 Vibrio分離來源：海洋生物、受污染海產(chǎn)品或臨床腹瀉患者標(biāo)本形態(tài)特征：革蘭氏陰性菌（G-），顯微鏡下呈多形性，主要為短桿狀、弧狀或逗點(diǎn)狀，無芽孢，具有極生單鞭毛，運(yùn)動極其活潑。核心科研與工業(yè)價(jià)值：該菌株是一株具有特定分子分型背景的副溶血性弧菌標(biāo)準(zhǔn)株。副溶血性弧菌是導(dǎo)致海產(chǎn)品源性感染、食物中毒以及急性胃腸炎的國際首要致病菌。該菌株廣泛應(yīng)用于食品安全致病菌定量質(zhì)控、藥敏檢測試驗(yàn)（AST）、新型致病機(jī)制研究、海水養(yǎng)殖魚蝦類病原學(xué)鑒定以及食品防腐劑效能評價(jià)。

2 核心生理生化特性與致病因子副溶血性弧菌 20130629002S01 表現(xiàn)出極其典型的嗜鹽性以及特定的致病特征：

核心嗜鹽特性：屬于高度專性嗜鹽性細(xì)菌。在完全不含氯化鈉（NaCl）的普通培養(yǎng)基中絕對無法生長。其生長所需的 NaCl 濃度范圍在 1% 至 8% 之間，最適生長氯化鈉濃度為 3%。致病因子鑒定表型：副溶血性弧菌的核心毒力因子包括耐熱溶血素（TDH）和/或 tdh 相關(guān)溶血素（TRH）。該菌株在日常質(zhì)控中通常通過神奈川現(xiàn)象（Kanagawa Phenomenon，KP）檢測試驗(yàn)（即在含高鹽和人紅細(xì)胞的 Wagatsuma 瓊脂平板上觀察是否產(chǎn)生特異性 beta-溶血環(huán)）或 PCR 擴(kuò)增 tdh/trh 基因來鎖定制定的毒力譜系。常規(guī)生化鑒定表型：氧化酶試驗(yàn)陽性；吲哚試驗(yàn)陽性；不發(fā)酵蔗糖，但發(fā)酵葡萄糖（不產(chǎn)氣）。在含有 3% NaCl 的三糖鐵（TSI）培養(yǎng)基中表現(xiàn)為底層變黃（產(chǎn)酸）、斜面不變色（不發(fā)酵乳糖/蔗糖）、不產(chǎn)硫化氫。

3 專用高鹽培養(yǎng)基配方規(guī)范

紅線警告：配置培養(yǎng)基時切勿漏加或少加氯化鈉。如果使用標(biāo)準(zhǔn)的普通營養(yǎng)肉湯（Nutrient Broth）或 LB 培養(yǎng)基，必須人為額外額外補(bǔ)加 3% 的 NaCl，否則該菌株將由于滲透壓失衡導(dǎo)致菌體裂解死亡。

A 3% NaCl 營養(yǎng)瓊脂平板/肉湯配方（用于日常擴(kuò)增、轉(zhuǎn)種與維持）基礎(chǔ)成分：牛肉膏 3.0 g，蛋白胨 10.0 g。關(guān)鍵嗜鹽添加劑：氯化鈉 NaCl 30.0 g（即 3% 終濃度）。瓊脂補(bǔ)劑（僅限平板固化）：瓊脂粉 15.0 g。溶劑：蒸餾水或去離子水 1000 mL。pH 調(diào)節(jié)：用 NaOH 調(diào)節(jié) pH 值至 8.0 到 8.5（該菌高度嗜堿，在酸性環(huán)境中極易死亡）。滅菌規(guī)范：121 ℃ 高壓蒸汽滅菌 15 分鐘。

B TCBS 瓊脂培養(yǎng)基配方（專性選擇性分離/鑒定平板）全稱：硫代硫酸鹽檸檬酸鹽膽鹽蔗糖瓊脂培養(yǎng)基（Thiosulfate Citrate Bile Salts Sucrose Agar）。培養(yǎng)基特征：由于副溶血性弧菌不發(fā)酵蔗糖，在該平板上生長 24 小時后，會形成直徑為 2 至 4 毫米、圓形、邊緣整齊、中心折光良好的藍(lán)綠色或綠色菌落（而發(fā)酵蔗糖的霍亂弧菌則呈現(xiàn)黃色菌落）。

C 菌株長期凍存保護(hù)液標(biāo)準(zhǔn)配方：含 3% NaCl 的營養(yǎng)肉湯 80% + 細(xì)胞/細(xì)菌級純甘油 20%。充分混勻后高壓滅菌，用于菌種深低溫保存。

4 物理環(huán)境控制參數(shù)培養(yǎng)溫度：37.0 ℃（最適生長溫度范圍 36.0 ℃ 至 38.0 ℃）氣相環(huán)境：常規(guī)常壓無菌空氣，需氧生長pH 范圍：8.0 - 8.5（最適堿性范圍）倍增速度：在營養(yǎng)充足、3% 鹽度且 pH 適宜的液體肉湯中，該菌分裂極快，代時可縮短至十幾分鐘，可在 6-8 小時內(nèi)使清亮的肉湯變?yōu)闃O度渾濁。

5 菌種活化、傳代操作規(guī)范傳代臨界點(diǎn)：固體平板接種后培養(yǎng) 18-24 小時，當(dāng)單個獨(dú)立菌落生長完全時，即可用于后續(xù)實(shí)驗(yàn)或轉(zhuǎn)種。切勿在 37 ℃ 下連續(xù)放置超過 48 小時，否則菌落會迅速進(jìn)入衰亡期并發(fā)生自溶。

標(biāo)準(zhǔn)平板劃線傳代步驟：1 從超低溫冰箱或冷凍干燥管中獲取菌種源。2 在超凈臺內(nèi)，點(diǎn)燃酒精燈。使用金屬接種環(huán)在火焰上燒灼至通紅，隨后在平板無菌區(qū)域邊緣貼靠冷卻。3 蘸取少許菌液或挑取單個獨(dú)立菌落，在 3% NaCl 營養(yǎng)瓊脂平板或 TCBS 平板上執(zhí)行標(biāo)準(zhǔn)三段或四段法劃線，以獲得稀釋的單菌落。4 劃線完畢后，蓋上皿蓋，將平板倒置（皿底朝上），放入 37 ℃ 恒溫培養(yǎng)箱中暗培養(yǎng) 18 到 24 小時。

6 冷凍干燥菌種管（安瓿管）的開啟與復(fù)蘇恢復(fù)流線1 準(zhǔn)備 1 支盛有 4.0 到 5.0 mL 預(yù)熱的 3% NaCl 營養(yǎng)肉湯管。2 拿起副溶血性弧菌 20130629002S01 凍干安瓿管，用 75% 酒精棉球擦拭其外壁進(jìn)行表面消毒。3 使用砂輪在安瓿管頂端（有棉簽一端）輕輕劃一圈細(xì)痕，隨后用一滴無菌水或微熱的玻璃棒觸碰劃痕處，使管壁產(chǎn)生微小裂紋。4 用無菌鑷子輕輕敲碎管帽，或用墊有無菌紗布的手指將管帽折斷。5 用無菌移液管吸取 0.5 mL 預(yù)熱的 3% NaCl 營養(yǎng)肉湯，緩緩注入安瓿管底部，輕柔吹吸數(shù)次，使干燥的菌體粉末完全溶解并呈均勻渾濁狀。6 吸出全部菌懸液，回注到剩余的 3% NaCl 營養(yǎng)肉湯管中，充分混勻。7 從中吸取 100 uL 菌液，接種到 3% NaCl 營養(yǎng)瓊脂平板或 TCBS 平板上進(jìn)行涂布/劃線，以確保獲取純凈的單菌落。8 將液體肉湯管與固體平板一并放入 37 ℃ 培養(yǎng)箱中培養(yǎng) 18-24 小時。

7 長期封存、生物安全與質(zhì)控規(guī)范生物安全級別：BSL-2（二級生物安全柜操作）。副溶血性弧菌屬于國家規(guī)定的二類或三類致病菌（視具體菌株毒力和感染途徑而定），日常操作必須在二級生物安全實(shí)驗(yàn)室內(nèi)進(jìn)行。所有接觸過該菌的槍頭、培養(yǎng)基、平板必須經(jīng)過 121 ℃ 高壓滅菌 30 分鐘后方可作為醫(yī)療廢物處理，嚴(yán)防污染外環(huán)境。超低溫長期保存：長期保存必須置于 -80 ℃ 超低溫冰箱（可維持 1-2 年）或永久存放于液氮罐（-196 ℃）中。嚴(yán)禁在 4 ℃ 普通冰箱中長期保存液體菌種，該菌在 4 ℃ 環(huán)境下極易發(fā)生“可活不可培養(yǎng)狀態(tài)”（VBNC）或直接大批自溶死亡。嗜鹽性與純度復(fù)核：每隔 5-10 代，應(yīng)將菌株同時接種至“不含 NaCl 的普通瓊脂平板”和“含 3% NaCl 的瓊脂平板”上。若不含鹽平板完全不生長，而高鹽平板生長出特征性邊緣整齊、灰白色、濕潤的單菌落，且革蘭氏染色顯示為陰性弧菌，方可確證該菌株未受到雜菌污染且嗜鹽表型完好。

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.