BioVector® Bacillus subtilis 3610 野生型枯草芽孢杆菌菌株 BioVector® Bacillus subtilis 3610 Wild-Type Strain

第一部分：中文说明

一、 产品基本信息与详细特征描述

• 产品名称：BioVector® Bacillus subtilis 3610 野生型枯草芽孢杆菌菌株

• 菌株名称：Bacillus subtilis 3610 (亦称 NCIB 3610)

• 物种来源：枯草芽孢杆菌 (Bacillus subtilis)

• 菌株属性：野生型分离株 (Wild-type isolate) / 模式研究菌株

• 生物安全级别：1级 (BSL-1)

• 详细特征描述：BioVector® Bacillus subtilis 3610 是一种极其经典的野生型枯草芽孢杆菌标准模式菌株。与实验室长期传代驯化、丧失了许多天然表型的传统高 competent 菌株（如 168 菌株）不同，3610 菌株完整地保留了野生型放线菌门/芽孢杆菌属的所有核心生理特性。该菌株具有强大的生物膜（Biofilm）形成能力、高效的群体运动能力（包括 Swarming 鞭毛群体滑行运动和 Swimming 游泳运动），以及复杂的细胞分化和自发成孢（Sporulation）机制。在固体培养基上，3610 菌株形成的菌落表面呈现出高度褶皱、干燥且具立体架构的复杂野生型特征，能分泌丰富的胞外多糖（EPS）和基质蛋白（如 TasA）。作为微生物社会性行为、生物膜发育、宿主-微生物相互作用（如植物根际益生菌定殖）等研究领域的金标准模式材料，该菌株在现代工程微生物学和发育生物学中占有举足轻重的地位。

二、 细胞培养条件与环境描述

• 常用培养基配方：为了维持菌株的正常生长或进行特定表型研究，可使用多种配方。常规克隆、扩增与维持选用标准 BioVector® LB 液体或固体琼脂培养基（每升包含：10g 胰蛋白胨，5g 酵母提取物，10g 氯化钠）。若进行标准的生物膜形成实验，强烈建议使用专门的 BioVector® MSgg 培养基（一种化学成分确定的最小培养基，能完美诱导其复杂的生物膜皱褶表型）。

• 培养环境参数：3610 菌株为专性需氧或兼性厌氧微生物，扩增培养时需置于恒温振荡培养箱中，标准气体环境为普通空气。培养温度设定为 37°C（进行生物膜静态诱导时通常可下调至 30°C 或 23°C）。液体震荡培养时，需维持每分钟 200 到 220 转的高转速，以保障充足的溶氧量。

三、 菌株复苏、扩增与表型验证操作步骤

1. 菌株快速复苏：从零下 80°C 超低温冰箱中取出 BioVector® Bacillus subtilis 3610 甘油冻存管，置于冰上略微解冻。在超净工作台内，用无菌接种环或微量移液枪吸取少量冻存菌液，划线接种于无抗生素的 BioVector® LB 固体琼脂平板上。

2. 平板孵育：将划线平板倒置放入 37°C 恒温培养箱中，孵育 12 到 16 小时（过夜）。次日即可观察到生长健壮、表面粗糙且边缘具扩展特性的单菌落。

3. 液体种子制备：挑取平板上典型的 3610 单菌落，接种至 5 毫升无菌的 BioVector® LB 液体培养基中。置于 37°C、每分钟 220 转的摇床中孵育 6 到 8 小时，直至菌液达到对数生长旺盛期（OD600 约 0.6 至 0.8），即可作为后续实验的种子液。

4. 野生型表型验证（群体运动实验）：为了验证 3610 菌株的野生型运动活力，可配制含有 0.7% 琼脂的 LB 平板（Swarming 实验）。将平板在超净台内完全风干，用微量移液器吸取 2 微里处于对数生长期末期的 3610 种子液，精准点样于平板正中央。静置待菌液完全吸附后，将平板正面朝上（勿倒置）放入 37°C 培养箱中孵育。数小时内即可观察到菌体自中心向外呈同心圆状或树突状快速自发滑行扩散，这是其野生型鞭毛运动能力的典型特征。

四、 菌株冻存与长期保藏技术

• 甘油菌种制备：将复苏纯化后的 3610 菌株在 LB 液体培养基中扩增至对数生长中期。吸取 700 微里新鲜菌液，与 300 微里无菌的 BioVector® 细胞级甘油（最终甘油体积百分比浓度为 30%）在无菌塑料冻存管中充分颠倒混匀，使其不留分层。

• 超低温长期保藏：将混合均匀的甘油冻存管直接移入零下 80°C 超低温冰箱中保存。该菌株具有极强的抗逆性和内生孢子形成能力，在甘油保护下可稳定保藏数年以上。取用时应严格遵循无菌原则，避免反复冻融。

五、 质量控制与科研应用指南

• 质量控制标准：BioVector® 发行的 Bacillus subtilis 3610 菌株经过严格的基因组及表型纯度鉴定。通过 16S rRNA 测序确认其物种唯一性；通过形态学观察确保其 100% 保持野生型高褶皱生物膜及 Swarming 运动特征，无向实验室退化型（平滑菌落、丧失生物膜）自发突变的污染；菌株经检测无任何外源噬菌体、杂菌污染。

• 核心实验应用方向：该野生型菌株广泛用于细菌生物膜（Biofilm）空间物理架构与细胞外基质基因调控研究、细菌群体运动（Swarming/Swimming）的流体力学与信号传导分析、芽孢杆菌孢子形成（Sporulation）及非对称细胞分裂的多级控制网络探索、植物根际促生菌（PGPR）对作物的定殖与生物防治机制研究，以及天然抗菌脂肽（如 Surfactin）的生物合成调控实验。

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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