BioVector? 副溶血性弧菌 RIMD 2210633 標準菌株——產(chǎn)品技術說明書

1 菌株基本信息與重大科研背景產(chǎn)品名稱：BioVector? 副溶血性弧菌 RIMD 2210633 標準菌株常用別名：Vibrio parahaemolyticus RIMD 2210633，RIMD2210633，副溶血弧菌大坂株生物學分類：細菌界 Bacteria / 變形菌門 Proteobacteria / 弧菌科 Vibrionaceae / 弧菌屬 Vibrio分離來源：1996年從日本大坂急性腸胃炎患者樣本中分離出的臨床大流行爆發(fā)株（血清型 O3:K6）形態(tài)特征：革蘭氏陰性（G-）多形性弧菌，呈短桿狀或單彎曲逗點狀，極端單鞭毛，運動能力極強。核心科研與國際里程碑價值：該菌株是全球首個完成全基因組測序的副溶血性弧菌標桿參考株（由日本研究團隊于2003年完成測序）?；蚪M包含兩條獨特的環(huán)狀染色體。它是國際上研究大流行 O3:K6 血清型弧菌毒力進化、三型分泌系統(tǒng)（TSS3-1 和 T6SS 等分泌系統(tǒng)）、環(huán)境適應機制、基因組島（Genomic Islands）以及全球食品安全分子流行病學溯源的公認第一標準底盤。

2 核心生理生化特性與三大致病系統(tǒng)RIMD 2210633 菌株作為測序標桿，其體內(nèi)蘊含著極其清晰且復雜的致病機制：

嚴格專性嗜鹽性：必須在含有氯化鈉 NaCl 的環(huán)境平衡下生存，最適 NaCl 生長濃度為 3%，完全無鹽環(huán)境下會迅速發(fā)生溶菌死亡。兩大核心毒力基因：該菌株在基因組上同時攜帶 tdh1 基因和 tdh2 基因，能夠大量高效地分泌耐熱溶血素（Thermostable Direct Hemolysin, TDH）。在神奈川現(xiàn)象（Kanagawa Phenomenon）測試中呈現(xiàn)強陽性（即在 Wagatsuma 瓊脂平板上使人紅細胞發(fā)生徹底的 beta-溶血）。雙三型分泌系統(tǒng)（T3SS1 / T3SS2）：T3SS1（位于染色體 I）：主要負責介導對宿主上皮細胞的細胞毒性，引發(fā)細胞自噬或凋亡。T3SS2（位于染色體 II）：主要介導腸道毒性，是導致臨床劇烈腹瀉、腸粘膜損傷及系統(tǒng)性腸炎的核心驅(qū)動力。

3 專用高鹽培養(yǎng)基配方規(guī)范

紅線警告：培養(yǎng)基配方必須人為額外補加 3% 的 NaCl，常規(guī)普通 LB 或普通營養(yǎng)肉湯無法維持該菌株存活。

A 3% NaCl 營養(yǎng)瓊脂平板/肉湯配方（用于日常日常傳代與常規(guī)擴增）基礎成分：牛肉膏 3.0 g，蛋白胨 10.0 g。核心專性添加劑：純氯化鈉 NaCl 30.0 g（即 3% 終濃度）。瓊脂添加（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)：使用高濃度 NaOH 調(diào)節(jié) pH 值至 8.0 到 8.5（該菌嗜堿怕酸）。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。生長表型：由于 RIMD 2210633 不具備發(fā)酵蔗糖的能力，在此平板上培養(yǎng) 24 小時后，會形成直徑為 2 至 5 毫米、圓形、表面濕潤、中心折光良好的藍綠色或深綠色菌落（借此可與霍亂弧菌等發(fā)酵蔗糖呈黃色的菌落進行明確區(qū)分）。

C 菌株長期凍存保護液標準配方：含 3% NaCl 的營養(yǎng)肉湯 80% + 細胞/細菌級純甘油 20%?；靹蚝蟾邏簻缇?，與對數(shù)生長期的菌液等體積混合后深低溫保存。

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：固體平板接種后培養(yǎng) 18-24 小時，當單個獨立菌落生長完全時，即可用于后續(xù)實驗或轉(zhuǎn)種。切勿在 37 ℃ 下連續(xù)放置超過 48 小時，否則菌落會迅速進入衰亡期并發(fā)生自溶。

標準平板劃線傳代步驟：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立菌落，在 3% NaCl 營養(yǎng)瓊脂平板或 TCBS 平板上執(zhí)行標準三段或四段法劃線，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）的開啟與復蘇恢復流線1 準備 1 支盛有 4.0 到 5.0 mL 預熱的 3% NaCl 營養(yǎng)肉湯管。2 拿起副溶血性弧菌 RIMD 2210633 凍干安瓿管，用 75% 酒精棉球擦拭其外壁進行表面消毒。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）。RIMD 2210633 為典型的高毒力臨床大流行株，日常實驗必須在具備二級生物安全防護屏障的實驗室內(nèi)進行。所有接觸過該菌的培養(yǎng)基、槍頭、移液管及平板必須經(jīng)過 121 ℃ 高壓滅菌至少 30 分鐘后方可廢棄，嚴防泄露污染環(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?；蚪M質(zhì)控復核（可選）：由于該菌株是全基因組測序的金標準，為了驗證實驗過程中未發(fā)生菌株混淆，可以定期提取該菌基因組 DNA，通過 PCR 特異性擴增其 T3SS1（如 vscC 基因）和 T3SS2（如 vscC2 基因）的標記性保守序列，雙重陽性且嗜鹽平板生長特征典型，方可確證其 RIMD 2210633 的本源血統(tǒng)。

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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