BioVector? 福賽坦納菌 ATCC 43037 標準菌株

BioVector? Tannerella forsythia ATCC 43037 Standard Strain

第一部分 中文說明

一 產品基本信息與遺傳學背景

• 菌株名稱：福賽坦納菌標準菌株（Tannerella forsythia，曾用名 Bacteroides forsythus）。

• 菌株編號與別名：ATCC 43037、JCM 10827、CIP 105218、CCUG 23111、DSM 102835。

• 物種分類：細菌界（Bacteria），擬桿菌門（Bacteroidota），擬桿菌綱（Bacteroidia），擬桿菌目（Bacteroidales），擬桿菌科（Bacteroidaceae），坦納菌屬（Tannerella）。

• 生物學與生理特異性特征：

  • 紅色復合體（Red Complex）核心成員：福賽坦納菌與牙齦卟啉單胞菌（P. gingivalis）和齒垢密螺旋體（T. denticola）共同構成了著名的口腔致病菌“紅色復合體”，是導致重度牙周炎、骨吸收和組織破壞的核心病原體。

  • 嚴格厭氧與專性營養(yǎng)依賴（N-乙酰胞壁酸依賴型）：ATCC 43037 是一種革蘭氏陰性（Gram-negative）、無動力、兩端呈尖細狀的梭形或短桿狀細菌。該菌具有極度苛刻的營養(yǎng)要求，其自身基因組缺乏合成 N-乙酰胞壁酸（NAM, N-acetylmuramic acid） 的關鍵酶，因此在人工培養(yǎng)時必須在培養(yǎng)基中額外添加外源性 NAM 才能生長。

• 來源歷史：最初由美國馬薩諸塞州波士頓的福賽斯牙科中心（Forsyth Dental Center）從人類高級牙周炎患者的牙齦下菌斑（Human subgingival plaque）中分離獲得，是全球廣泛接受的福賽坦納菌模式參考菌株（Type strain）。

• 生物安全級別：2級（BSL-2）。涉及活菌的操作必須在二級生物安全柜內進行。

二 核心科研價值與轉化醫(yī)學應用

福賽坦納菌是國際口腔微生物學界公認最難培養(yǎng)但極具臨床意義的病原菌之一：

1. 牙周病學與骨吸收機制研究（Periodontology & Bone Resorption）：廣泛用于研究其特征性的外膜蛋白（表面 S 層蛋白 / S-layer proteins）、BspA 蛋白（富含亮氨酸的重復序列蛋白）如何誘導宿主上皮細胞分泌促炎細胞因子（IL-6, TNF-alpha），以及如何激活破骨細胞導致牙槽骨吸收。

2. 口腔多物種生物膜模型（Multi-species Biofilm Modeling）：在體外構建“紅色復合體”或多菌種人工牙斑塊（Biofilm）模型時，ATCC 43037 作為核心組分，用于分析其與其他口腔共生菌/致病菌（如宿主相互依賴的協(xié)同作用）的共凝集及空間分布特征。

3. 診斷檢測試劑驗證（Metrology & Molecular Diagnostics）：作為法定的模式菌株，廣泛用于各種口腔微生物 PCR 檢測試劑盒、高通量測序（NGS）宏基因組學分析以及臨床厭氧菌鑒定系統(tǒng)的特異性對照。

三 實驗室菌株復蘇、擴增傳代與冷凍保存標準步驟

1. 專用培養(yǎng)基與嚴格環(huán)境配置

福賽坦納菌對環(huán)境極其敏感，接觸氧氣會迅速失活：

• 核心專用培養(yǎng)基配方（NAM 補充型血瓊脂 / 肉湯）：

  • 基礎：大豆酪蛋白消化物（TSB/TSA）或腦心浸液（BHI）培養(yǎng)基，補充 5% 滅菌脫纖維羊血或馬血、0.5% 酵母浸膏（Yeast Extract）、氯化血紅素（Hemin，5 microgram/mL）和維生素 K1（1 microgram/mL）。

  • 關鍵添加劑：必須在無菌條件下加入經微孔濾膜過濾除菌的 N-乙酰胞壁酸（NAM），其最終工作濃度必須達到 10 microgram/mL 至 100 microgram/mL（常用 10-20 microgram/mL）。

• 培養(yǎng)環(huán)境參數(shù)：溫度為 37 攝氏度，必須置于嚴格厭氧環(huán)境（無氧加濕環(huán)境，如：85% $N_2$ + 10% $CO_2$ + 5% $H_2$）的厭氧培養(yǎng)箱或使用高效厭氧產氣包的厭氧罐中孵育。

2. 凍干粉/凍存菌種復蘇步驟（Thawing & Revitalization）

1. 準備工作：將補充有 NAM、血紅素和維生素 K1 的固體血瓊脂平板或液體肉湯提前置于厭氧箱中進行預還原（Pre-reduction，至少 12-24 小時），以徹底驅除培養(yǎng)基內的游離氧。

2. 在生物安全柜內小心打開含有 ATCC 43037 的凍干安瓿管。

3. 用無菌移液管吸取約 0.5 mL 預還原的厭氧肉湯，滴加到凍干菌塊上使其完全溶解。

4. 將全量菌懸液迅速接種到預還原的 NAM 補充型血瓊脂平板上（進行密集區(qū)域密集劃線，由于其生長微弱，早期建議集中接種）。

5. 極重要：接種后立即將平板送回 37 攝氏度嚴格厭氧箱 中。由于福賽坦納菌生長速度極慢，通常需要連續(xù)孵育 5 至 7 天（甚至更久）方可看到針尖大小的微小菌落。

3. 菌株傳代與日常維持（Passaging & Maintenance）

• 形態(tài)觀察：在固體平板上，ATCC 43037 形成極微小、凸起、光滑、微白至半透明的菌落。

• 傳代操作：挑取平板上生長良好的微小菌落，接種至新鮮的預還原 NAM 補充型液體肉湯或固體平板上。液體培養(yǎng)通常需要 3 至 5 天方可達到對數(shù)生長晚期。建議每次傳代時間控制在菌落剛長出的活躍期，嚴禁使用干枯或長期暴露在室溫環(huán)境下的老舊平板進行轉接。

4. 菌株長期冷凍保存（Cryopreservation）

• 長期甘油冷凍法：采集處于對數(shù)生長活躍期的液體培養(yǎng)物。在嚴格厭氧環(huán)境下，按照 80% 液體菌懸液 + 20% 滅菌高純度甘油 的比例在凍存管中輕柔混勻，或使用含 15% 甘油的預還原厭氧肉湯。

• 保存條件：混勻后，立即將凍存管移出并投入 -80 攝氏度 超低溫冰箱，或者直接放入 液氮（-196 攝氏度） 中。在連續(xù)穩(wěn)定的超低溫下，可實現(xiàn)數(shù)年以上的無限期穩(wěn)定保存。

Part 2 English Section

I General Information and Genetic Architecture

• Organism Name: Tannerella forsythia Standard Reference / Type Strain (formerly known as Bacteroides forsythus).

• Strain Designations and Aliases: ATCC 43037, JCM 10827, CIP 105218, CCUG 23111, DSM 102835.

• Taxonomic Classification: Domain Bacteria, Phylum Bacteroidota, Class Bacteroidia, Order Bacteroidales, Family Bacteroidaceae, Genus Tannerella.

• Biological and Physiological Core Framework:

  • Red Complex Cornerstone: Along with Porphyromonas gingivalis and Treponema denticola, Tannerella forsythia belongs to the infamous "Red Complex" group of oral anaerobic pathogens, globally recognized as the main clinical drivers of advanced periodontitis, tissue loss, and alveolar bone destruction.

  • Strict Anaerobe & Auxotrophic Requirement (N-Acetylmuramic Acid Dependent): ATCC 43037 is a Gram-negative, non-motile, spindle-shaped or fusiform rod-like bacillus with tapered ends. This species exhibits an extremely fastidious nutritional profile; its genome lacks the essential biosynthetic machinery to produce N-acetylmuramic acid (NAM), a vital element for cell wall peptidoglycan assembly. Consequently, it absolutely requires exogenous NAM supplementation to proliferate in artificial media.

• Source Isolation History: Originally recovered and isolated from human subgingival plaque samples harvested from patients diagnosed with severe periodontitis at the Forsyth Dental Center in Boston, Massachusetts, USA. It serves as the official international taxonomic reference type strain for the species.

• Biosafety Matrix: Classified under Biosafety Level 2 (BSL-2) containment parameters. All workflows with viable cultures require certified biosafety enclosures and anaerobically controlled systems.

II Strategic Research Value and Translational Fields

Tannerella forsythia is universally regarded as one of the most fastidious yet pathologically significant oral microbes:

1. Periodontitis Etiology and Alveolar Bone Resorption: Widely used to characterize its unique outer cell surface layer (S-layer proteins) and the BspA virulence factor (a leucine-rich repeat protein). Researchers assess how these surface components trigger host epithelial arrays to secrete pro-inflammatory cytokines (such as IL-6 and TNF-alpha) and drive osteoclast differentiation.

2. Polymicrobial Oral Biofilm Architecture: Deployed as a key component in multi-species oral biofilm platforms (mimicking the Red Complex environment), illuminating spatial arrangement patterns, co-aggregation arrays, and synergistic nutritional interdependencies with other oral pathobionts.

3. Diagnostic Validation and Baseline Metrology: Serves as the definitive global reference control strain to benchmark the analytical sensitivity, target specificity, and alignment metrics of novel periodontal PCR diagnostic arrays, microfluidic chips, and next-generation sequencing (NGS) metagenomic profiling platforms.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Medium Formulations and Controlled Ambient Specifications

This strict anaerobe is highly sensitive to trace oxygen; brief atmospheric exposure during active growth can cause irreversible loss of viability:

• Specialized Enriched Growth Media (NAM-Fortified Blood Agar/Broth):

  • Basal Matrix: Premium Tryptic Soy Broth/Agar (TSB/TSA) or Brain Heart Infusion (BHI) matrix, fortified with 5% defibrinated sheep or horse blood, 0.5% yeast extract, hemin (5 microgram/mL), and coenzyme vitamin K1 (1 microgram/mL).

  • Critical Nutrient Additive: Supplement the media sterilely with filter-sterilized N-acetylmuramic acid (NAM) to achieve a definitive final work concentration of 10 microgram/mL to 100 microgram/mL (typically optimized at 10–20 microgram/mL).

• Atmospheric Parameters: Regulated strictly at 37 degrees Celsius within a rigidly anaerobic workspace (e.g., 85% $N_2$ + 10% $CO_2$ + 5% $H_2$) or inside specialized anaerobic jars configured with palladium catalysts and active gas-scavenging pouches.

2. Thawing and Revitalization Routine (Lyophilized Pellet Revitalization)

1. Pre-reduction Workflow: Solid blood agar plates and liquid media supplemented with NAM must be placed inside the anaerobic workstation for pre-reduction (minimum 12–24 hours) prior to inoculation to deplete dissolved residual oxygen within the media matrix.

2. Inside an approved biosafety enclosure, open the sealed outer glass ampoule containing the lyophilized ATCC 43037 pellet.

3. Utilizing a sterile pipette, deliver approximately 0.5 mL of the pre-reduced anaerobic broth onto the pellet. Mix gently until completely dissolved.

4. Promptly transfer the suspension onto the pre-reduced NAM-fortified blood agar plates, using a dense quadrant streak configuration to concentrate cell density.

5. Critical Instruction: Immediately return the inoculated plates to the 37 degrees Celsius strict anaerobic workspace. Because this strain grows very slowly, initial pinpoint colonies typically emerge only after 5 to 7 days of continuous incubation.

3. Subculturing and Routine Maintenance

• Colony Morphology: On solid matrices, ATCC 43037 colonies present as small, smooth, convex, translucent-to-white pinpoint dots.

• Passaging Routine: Pick distinct colonies using a sterile loop and re-streak onto fresh pre-reduced solid NAM-fortified media or inoculate into liquid broth arrays. Liquid cultures typically require 3 to 5 days to reach stable late-log phase density. Subculturing must be performed using active, freshly emerged colonies; do not attempt to transfer cultures from aged, desiccated plates or plates left at room temperature.

4. Cryopreservation Protocol

• Long-Term Cryo-Freezing Routine: Harvest active liquid cultures during their peak logarithmic phase. Under anaerobic conditions, blend the cell suspension in a sterile cryovial filled with 80% active culture slurry and 20% sterile high-purity glycerol (or utilize a 15% glycerol-fortified pre-reduced anaerobic broth matrix).

• Storage Configuration: Mix thoroughly, extract from the anaerobic hood, and flash-freeze instantly at -80 degrees Celsius or drop directly into liquid nitrogen (-196 degrees Celsius) for indefinite functional preservation across decades.

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