BioVector? Bacillus subtilis 3610 野生型枯草芽孢桿菌菌株 BioVector? Bacillus subtilis 3610 Wild-Type Strain

第一部分：中文說(shuō)明

一、 產(chǎn)品基本信息與詳細(xì)特征描述

• 產(chǎn)品名稱：BioVector? Bacillus subtilis 3610 野生型枯草芽孢桿菌菌株

• 菌株名稱：Bacillus subtilis 3610 (亦稱 NCIB 3610)

• 物種來(lái)源：枯草芽孢桿菌 (Bacillus subtilis)

• 菌株屬性：野生型分離株 (Wild-type isolate) / 模式研究菌株

• 生物安全級(jí)別：1級(jí) (BSL-1)

• 詳細(xì)特征描述：BioVector? Bacillus subtilis 3610 是一種極其經(jīng)典的野生型枯草芽孢桿菌標(biāo)準(zhǔn)模式菌株。與實(shí)驗(yàn)室長(zhǎng)期傳代馴化、喪失了許多天然表型的傳統(tǒng)高 competent 菌株（如 168 菌株）不同，3610 菌株完整地保留了野生型放線菌門/芽孢桿菌屬的所有核心生理特性。該菌株具有強(qiáng)大的生物膜（Biofilm）形成能力、高效的群體運(yùn)動(dòng)能力（包括 Swarming 鞭毛群體滑行運(yùn)動(dòng)和 Swimming 游泳運(yùn)動(dòng)），以及復(fù)雜的細(xì)胞分化和自發(fā)成孢（Sporulation）機(jī)制。在固體培養(yǎng)基上，3610 菌株形成的菌落表面呈現(xiàn)出高度褶皺、干燥且具立體架構(gòu)的復(fù)雜野生型特征，能分泌豐富的胞外多糖（EPS）和基質(zhì)蛋白（如 TasA）。作為微生物社會(huì)性行為、生物膜發(fā)育、宿主-微生物相互作用（如植物根際益生菌定殖）等研究領(lǐng)域的金標(biāo)準(zhǔn)模式材料，該菌株在現(xiàn)代工程微生物學(xué)和發(fā)育生物學(xué)中占有舉足輕重的地位。

二、 細(xì)胞培養(yǎng)條件與環(huán)境描述

• 常用培養(yǎng)基配方：為了維持菌株的正常生長(zhǎng)或進(jìn)行特定表型研究，可使用多種配方。常規(guī)克隆、擴(kuò)增與維持選用標(biāo)準(zhǔn) BioVector? LB 液體或固體瓊脂培養(yǎng)基（每升包含：10g 胰蛋白胨，5g 酵母提取物，10g 氯化鈉）。若進(jìn)行標(biāo)準(zhǔn)的生物膜形成實(shí)驗(yàn)，強(qiáng)烈建議使用專門的 BioVector? MSgg 培養(yǎng)基（一種化學(xué)成分確定的最小培養(yǎng)基，能完美誘導(dǎo)其復(fù)雜的生物膜皺褶表型）。

• 培養(yǎng)環(huán)境參數(shù)：3610 菌株為專性需氧或兼性厭氧微生物，擴(kuò)增培養(yǎng)時(shí)需置于恒溫振蕩培養(yǎng)箱中，標(biāo)準(zhǔn)氣體環(huán)境為普通空氣。培養(yǎng)溫度設(shè)定為 37°C（進(jìn)行生物膜靜態(tài)誘導(dǎo)時(shí)通?？上抡{(diào)至 30°C 或 23°C）。液體震蕩培養(yǎng)時(shí)，需維持每分鐘 200 到 220 轉(zhuǎn)的高轉(zhuǎn)速，以保障充足的溶氧量。

三、 菌株復(fù)蘇、擴(kuò)增與表型驗(yàn)證操作步驟

1. 菌株快速?gòu)?fù)蘇：從零下 80°C 超低溫冰箱中取出 BioVector? Bacillus subtilis 3610 甘油凍存管，置于冰上略微解凍。在超凈工作臺(tái)內(nèi)，用無(wú)菌接種環(huán)或微量移液槍吸取少量?jī)龃婢?，劃線接種于無(wú)抗生素的 BioVector? LB 固體瓊脂平板上。

2. 平板孵育：將劃線平板倒置放入 37°C 恒溫培養(yǎng)箱中，孵育 12 到 16 小時(shí)（過(guò)夜）。次日即可觀察到生長(zhǎng)健壯、表面粗糙且邊緣具擴(kuò)展特性的單菌落。

3. 液體種子制備：挑取平板上典型的 3610 單菌落，接種至 5 毫升無(wú)菌的 BioVector? LB 液體培養(yǎng)基中。置于 37°C、每分鐘 220 轉(zhuǎn)的搖床中孵育 6 到 8 小時(shí)，直至菌液達(dá)到對(duì)數(shù)生長(zhǎng)旺盛期（OD600 約 0.6 至 0.8），即可作為后續(xù)實(shí)驗(yàn)的種子液。

4. 野生型表型驗(yàn)證（群體運(yùn)動(dòng)實(shí)驗(yàn)）：為了驗(yàn)證 3610 菌株的野生型運(yùn)動(dòng)活力，可配制含有 0.7% 瓊脂的 LB 平板（Swarming 實(shí)驗(yàn)）。將平板在超凈臺(tái)內(nèi)完全風(fēng)干，用微量移液器吸取 2 微里處于對(duì)數(shù)生長(zhǎng)期末期的 3610 種子液，精準(zhǔn)點(diǎn)樣于平板正中央。靜置待菌液完全吸附后，將平板正面朝上（勿倒置）放入 37°C 培養(yǎng)箱中孵育。數(shù)小時(shí)內(nèi)即可觀察到菌體自中心向外呈同心圓狀或樹(shù)突狀快速自發(fā)滑行擴(kuò)散，這是其野生型鞭毛運(yùn)動(dòng)能力的典型特征。

四、 菌株凍存與長(zhǎng)期保藏技術(shù)

• 甘油菌種制備：將復(fù)蘇純化后的 3610 菌株在 LB 液體培養(yǎng)基中擴(kuò)增至對(duì)數(shù)生長(zhǎng)中期。吸取 700 微里新鮮菌液，與 300 微里無(wú)菌的 BioVector? 細(xì)胞級(jí)甘油（最終甘油體積百分比濃度為 30%）在無(wú)菌塑料凍存管中充分顛倒混勻，使其不留分層。

• 超低溫長(zhǎng)期保藏：將混合均勻的甘油凍存管直接移入零下 80°C 超低溫冰箱中保存。該菌株具有極強(qiáng)的抗逆性和內(nèi)生孢子形成能力，在甘油保護(hù)下可穩(wěn)定保藏?cái)?shù)年以上。取用時(shí)應(yīng)嚴(yán)格遵循無(wú)菌原則，避免反復(fù)凍融。

五、 質(zhì)量控制與科研應(yīng)用指南

• 質(zhì)量控制標(biāo)準(zhǔn)：BioVector? 發(fā)行的 Bacillus subtilis 3610 菌株經(jīng)過(guò)嚴(yán)格的基因組及表型純度鑒定。通過(guò) 16S rRNA 測(cè)序確認(rèn)其物種唯一性；通過(guò)形態(tài)學(xué)觀察確保其 100% 保持野生型高褶皺生物膜及 Swarming 運(yùn)動(dòng)特征，無(wú)向?qū)嶒?yàn)室退化型（平滑菌落、喪失生物膜）自發(fā)突變的污染；菌株經(jīng)檢測(cè)無(wú)任何外源噬菌體、雜菌污染。

• 核心實(shí)驗(yàn)應(yīng)用方向：該野生型菌株廣泛用于細(xì)菌生物膜（Biofilm）空間物理架構(gòu)與細(xì)胞外基質(zhì)基因調(diào)控研究、細(xì)菌群體運(yùn)動(dòng)（Swarming/Swimming）的流體力學(xué)與信號(hào)傳導(dǎo)分析、芽孢桿菌孢子形成（Sporulation）及非對(duì)稱細(xì)胞分裂的多級(jí)控制網(wǎng)絡(luò)探索、植物根際促生菌（PGPR）對(duì)作物的定殖與生物防治機(jī)制研究，以及天然抗菌脂肽（如 Surfactin）的生物合成調(diào)控實(shí)驗(yàn)。

PART 2: ENGLISH SECTION

I. General Information and Detailed Product Characterization

• Product Name: BioVector? Bacillus subtilis 3610 Wild-Type Strain

• Strain Name: Bacillus subtilis 3610 (also designated as NCIB 3610)

• Organism: Bacillus subtilis

• Strain Property: Wild-type isolate / Standard model strain

• Biosafety Level: BSL-1

• Detailed Description: BioVector? Bacillus subtilis 3610 is an iconic and highly indispensable wild-type isolate widely recognized as the gold standard model strain for researching non-domesticated bacterial behaviors. In sharp contrast to highly domesticated, highly competent laboratory lineages (such as strain 168) which have accumulated mutations resulting in the loss of ancestral multicellular phenotypes during decades of laboratory passaging, strain 3610 retains the complete repertoire of natural physiological hallmarks characteristic of the genus Bacillus. It exhibits robust biofilm development profiles, high-velocity multicellular motility behaviors (including flagella-driven swarming on surfaces and swimming in liquids), and highly coordinated cell differentiation and spontaneous sporulation pathways. When grown on solid agar, 3610 gives rise to visually striking, complex colonies characterized by an intricately wrinkled, dry, and highly structured architectural morphology, supported by the intensive secretion of extracellular polysaccharides (EPS) and matrix proteins (such as TasA). Serving as a foundational matrix for studying socio-microbiological interactions, biofilm kinetics, and host-microbe ecology (such as plant-rhizosphere colonization), this strain represents a core molecular tool in modern microbiology and developmental biology.

II. Culture Conditions and Environmental Parameters

• Recommended Medium Formulations: Depending on experimental benchmarks, various media can be implemented. For routine preservation, propagation, and cloning assays, standard BioVector? LB Liquid Medium or Solid Agar Medium (10g/L Tryptone, 5g/L Yeast Extract, 10g/L NaCl) is highly effective. To evaluate and induce premium, highly structured biofilm architecture, the utilizing of specialized BioVector? MSgg Medium (a chemically defined minimal media optimized for robust architectural wrinkling phenotypes) is strictly recommended.

• Incubation Parameters: As an obligate aerobe or facultative anaerobe, strain 3610 must be incubated under regular atmospheric air inputs. The benchmark cultivation temperature is set at 37°C (though temperature inputs are frequently lowered to 30°C or 23°C during static biofilm pellicle assays). For liquid propagation, maintain a high agitation rate spanning 200 to 220 RPM in a shaking incubator to ensure maximum dissolved oxygen availability.

III. Thawing, Propagation, and Phenotypic Validation Protocol

1. Rapid Strain Thawing: Retrieve the BioVector? Bacillus subtilis 3610 glycerol stock from the minus 80°C ultra-low freezer and place it immediately on ice to thaw partially. Within a sterile biosafety cabinet, use a sterile inoculation loop or micropipette to transfer a small aliquot of the frozen culture onto a selective-antibiotic-free BioVector? LB Agar plate, executing a standard quadrant streak.

2. Plate Incubation: Invert the streaked agar plate and place it inside a 37°C incubator for 12 to 16 hours (overnight). Distinct, robust single colonies demonstrating characteristically matte, rough, and spreading edges will emerge the following day.

3. Liquid Starter Preparation: Pick a representative single colony displaying the classic wrinkled phenotype from the plate and inoculate it into 5 milliliters of sterile BioVector? LB Liquid Medium. Incubate in a shaking incubator at 37°C at 220 RPM for 6 to 8 hours until the biomass enters its mid-exponential phase (OD600 approximately 0.6–0.8) to establish the working starter culture.

4. Phenotypic Validation (Surface Swarming Assay): To verify the functional wild-type motility of the 3610 stock, prepare freshly poured LB Agar plates containing precisely 0.7% agar. Allow the plates to dry completely under the laminar flow hood. Inoculate 2 microliters of the exponential starter culture precisely onto the center of the agar surface. Allow the droplet to fully absorb into the agar, then incubate the plate facing right side up (do not invert) inside a 37°C incubator. Within a few hours, massive coordinated flagellar migration will be visible as a rapid, concentric or dendritic expansion radiating outward from the center point, validating wild-type motility integrity.

IV. Strain Preservation and Long-Term Storage Methodology

• Glycerol Stock Formulation: Propagate a verified 3610 culture in LB liquid medium until it reaches its mid-logarithmic developmental window. Combine 700 microliters of this fresh bacterial culture thoroughly with 300 microliters of sterile, BioVector? Cell-Grade Glycerol to achieve a final concentration of 30% glycerol inside a sterile cryogenic vial, ensuring a homogeneous distribution.

• Ultra-Low Temperature Archiving: Store the formulated cryovials directly within a minus 80°C ultra-low temperature freezer. Owing to its natural capacity for resilient endospore formation and glycerol cryoprotection, the strain can be stably archived for multiple years. Always extract samples using aseptic protocols and avoid repetitive thermal fluctuations.

V. Quality Control and Research Application Guidelines

• Quality Control Standards: The BioVector? Bacillus subtilis 3610 strain undergoes rigorous genetic identity and phenotypic purity validation. Full-length 16S rRNA gene sequencing certifies absolute species alignment. Phenotypic profiling guarantees 100% preservation of highly wrinkled biofilm matrices and swarming motility kinetics, ensuring no contamination from domesticated spontaneous mutants that produce smooth, flat colonies. The final lot is certified free from exogenous bacteriophages or symbiotic contaminants.

• Core Experimental Applications: This pristine wild-type strain is heavily utilized for investigating the spatial physics and genetic networks governing bacterial biofilm and pellicle matrix development, hydrodynamic and signaling tracking during multicellular swarming/swimming behaviors, multi-tiered transcriptional regulatory networks driving asymmetric cell division and sporulation, molecular dialogues optimizing plant growth-promoting rhizobacteria (PGPR) colonization and biocontrol dynamics, and biosynthetic scaling of natural lipopeptide surfactants (such as Surfactin).

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