Key Experimental Techniques in Biotechnology | 生物技术实验考点解析

📚 Key Experimental Techniques in Biotechnology | 生物技术实验考点解析

Biotechnology practicals are a core part of A-Level Biology, testing your ability to handle microorganisms, enzymes, DNA and proteins with precision. This article breaks down the most frequently examined experimental techniques, the underlying principles, and the common pitfalls you must avoid.

生物技术实验是 A-Level 生物学的核心内容，考查你精确处理微生物、酶、DNA 和蛋白质的能力。本文系统梳理最高频的实验考点、背后的原理以及你必须避免的常见错误。

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1. Aseptic Technique | 无菌技术

The central aim of aseptic technique is to prevent contamination of pure cultures by unwanted microorganisms. This involves sterilising equipment, working near a Bunsen burner flame, and using sterile pipettes and loops.

无菌技术的核心目的是防止纯培养物被杂菌污染。具体操作包括对器材进行灭菌、在酒精灯火焰附近操作，以及使用无菌移液管和接种环。

• Sterilise the inoculating loop by heating it to red hot before and after transfer.

  接种环在使用前后都要加热至红热以彻底灭菌。

• Pass the neck of the culture bottle through the flame to create an updraft that prevents airborne microbes from entering.

  培养瓶瓶口需快速通过火焰，利用上升气流防止空气中微生物进入。

• Work in a laminar flow cabinet or near a flame to reduce the risk of contamination.

  应在超净工作台或火焰附近操作，以降低污染风险。

Common exam question: Why must the lid of the Petri dish be held over the plate, not placed on the bench? Because it protects the agar surface from airborne contamination.

常见考点：为什么培养皿的盖子要悬在皿口上方而不是放在台面上？答：防止空气中的微生物污染琼脂表面。

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2. Preparation of Culture Media | 培养基的制备

Culture media provide nutrients, energy sources, pH buffers and sometimes selective agents for microbial growth. The two main forms are liquid broth and solid agar plates.

培养基为微生物提供营养物质、能量来源、pH 缓冲剂，有时还包含选择剂。两种主要形式是液体肉汤和固体琼脂平板。

• Agar is a polysaccharide extracted from seaweed; it solidifies at about 45 °C and melts at about 95 °C, so it remains solid at typical incubation temperatures.

  琼脂是从海藻中提取的多糖，在约 45 °C 凝固，约 95 °C 融化，因此在通常的培养温度下能保持固态。

• Nutrient broth contains a carbon source (e.g. glucose), a nitrogen source (e.g. peptone), mineral ions and vitamins.

  营养肉汤含有碳源（如葡萄糖）、氮源（如蛋白胨）、无机盐离子和维生素。

• Selective media contain antibiotics or other agents that allow only specific organisms to grow, e.g. MacConkey agar selects for Gram-negative bacteria.

  选择培养基含有抗生素或其他物质，只允许特定微生物生长，例如 MacConkey 琼脂可选择革兰氏阴性菌。

Number of colonies = Number of viable cells × Dilution factor

菌落数 = 活菌数 × 稀释倍数

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3. Serial Dilution and Spread Plating | 系列稀释与涂布平板法

Serial dilution is used to reduce the concentration of bacteria so that individual colonies can be counted. A known volume of diluted culture is spread over the surface of an agar plate.

系列稀释用于降低细菌浓度，使单个菌落可以被计数。将已知体积的稀释培养液涂布在琼脂平板表面。

• Take 1 cm³ of original culture and add to 9 cm³ of sterile water; this gives a 10⁻¹ dilution. Repeat to obtain 10⁻², 10⁻³, etc.

  取 1 cm³ 原始培养液加入 9 cm³ 无菌水，得到 10⁻¹ 稀释液；重复操作可得到 10⁻²、10⁻³ 等梯度稀释液。

• Use a sterile spreader to distribute the sample evenly over the surface.

  使用无菌涂布器将样品均匀涂布在培养基表面。

• Only plates with between 30 and 300 colonies are suitable for counting; fewer may be unreliable, and more may be too crowded to count.

  只有菌落数在 30 到 300 之间的平板适合计数；太少不可靠，太多则拥挤难以计数。

Always state the dilution factor when calculating the original cell density. For example, if 0.1 cm³ of a 10⁻⁶ dilution gives 45 colonies, the original cell count is 45 × 10⁶ × 10 = 4.5 × 10⁸ CFU cm⁻³.

计算原始细胞密度时一定要说明稀释倍数。例如，0.1 cm³ 的 10⁻⁶ 稀释液长出 45 个菌落，则原始细胞数 = 45 × 10⁶ × 10 = 4.5 × 10⁸ CFU cm⁻³。

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4. Colony Counting and Viable Cell Count | 菌落计数与活菌计数

The number of colonies on a plate represents the number of viable cells in the original sample, assuming each colony arises from one cell. This method is called a viable cell count.

平板上的菌落数代表原始样品中的活菌数，前提是每个菌落由一个细胞形成。这种方法称为活菌计数。

• Only live cells form colonies; dead cells are not counted.

  只有活细胞才能形成菌落，死细胞不会被计数。

• To increase reliability, count plates from several dilutions and take an average.

  为提高可靠性，应计数多个稀释度的平板并取平均值。

• Express results as colony-forming units per cm³ (CFU cm⁻³), because one colony may originate from a clump of cells.

  结果用每立方厘米的菌落形成单位（CFU cm⁻³）表示，因为一个菌落可能来源于一团细胞。

Exam tip: Always distinguish between total cell count (including dead cells, using a haemocytometer) and viable cell count (using pour plates or spread plates).

考试提示：务必区分总细胞计数（含死细胞，用血球计数板）和活菌计数（用浇板法或涂布法）。

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5. Gram Staining | 革兰氏染色

Gram staining differentiates bacteria into Gram-positive and Gram-negative based on the structure of their cell walls.

革兰氏染色根据细胞壁结构将细菌分为革兰氏阳性和革兰氏阴性。

• Apply crystal violet, then iodine (mordant), then alcohol (decolouriser), then safranin (counterstain).

  依次添加结晶紫、碘液（媒染剂）、酒精（脱色剂）和番红（复染剂）。

• Gram-positive bacteria have a thick peptidoglycan layer that retains crystal violet, appearing purple.

  革兰氏阳性菌具有较厚的肽聚糖层，能保留结晶紫，呈紫色。

• Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane; alcohol removes the crystal violet, and they take up safranin, appearing pink/red.

  革兰氏阴性菌肽聚糖层薄且有外膜；酒精洗去结晶紫后，菌体吸收番红，呈粉红色或红色。

Crystal violet → Iodine → Alcohol → Safranin

结晶紫 → 碘液 → 酒精 → 番红

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6. Enzyme-Linked Immunosorbent Assay (ELISA) | 酶联免疫吸附测定

ELISA is used to detect the presence of antigens or antibodies in a sample. It relies on the specificity of antigen-antibody interactions and an enzyme-linked antibody that produces a colour change.

ELISA 用于检测样品中是否存在抗原或抗体。它依赖抗原-抗体反应的特异性，以及酶标抗体催化底物产生颜色变化。

• In a direct ELISA, antigen is immobilised on a well, a primary antibody binds, then an enzyme-linked secondary antibody binds and converts a colourless substrate into a coloured product.

  在直接 ELISA 中，抗原固定在孔内，一抗与之结合，然后酶标二抗结合并催化无色底物变为有色产物。

• In a sandwich ELISA, the antigen is captured between two antibodies, increasing specificity.

  在夹心 ELISA 中，抗原被两层抗体夹在中间，特异性更高。

• The intensity of colour is proportional to the amount of antigen present; it can be measured with a colorimeter.

  颜色深浅与抗原量成正比，可用比色计测量。

Common error: Students forget to wash the wells between steps; washing removes unbound antibodies and reduces false positives.

常见错误：学生忘记在每步之间洗涤孔板；洗涤可以去除未结合的抗体，降低假阳性。

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7. Gel Electrophoresis | 凝胶电泳

Gel electrophoresis separates DNA fragments or proteins based on their size and charge. DNA is negatively charged, so it moves towards the positive electrode.

凝胶电泳根据分子大小和电荷分离 DNA 片段或蛋白质。DNA 带负电荷，因此向正极移动。

• Agarose gel is used for DNA; polyacrylamide gel is often used for proteins.

  DNA 常用琼脂糖凝胶；蛋白质常用聚丙烯酰胺凝胶。

• Smaller fragments move faster and travel further than larger fragments.

  小片段比大片段移动更快、迁移更远。

• A DNA ladder (known size markers) is loaded into one well to estimate the size of unknown fragments.

  将已知大小的 DNA marker 加入一个孔中，用于估算未知片段的大小。

For protein separation under denaturing conditions, SDS is added to give all proteins a uniform negative charge per unit mass, so separation is based purely on molecular weight.

在变性条件下分离蛋白质时，加入 SDS 使所有蛋白质每单位质量带有均匀的负电荷，因此分离仅依赖分子量。

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8. Polymerase Chain Reaction (PCR) | 聚合酶链式反应

PCR amplifies a specific DNA region exponentially. It requires a DNA template, primers, free nucleotides, and a heat-stable DNA polymerase (Taq polymerase).

PCR 以指数方式扩增特定 DNA 区域。需要 DNA 模板、引物、游离核苷酸和耐热 DNA 聚合酶（Taq 聚合酶）。

Stage	Temperature	Purpose
Denaturation	95 °C	Hydrogen bonds between DNA strands break.
Annealing	50–65 °C	Primers bind to complementary sequences.
Extension	72 °C	Taq polymerase adds nucleotides.

PCR 循环：变性 95 °C，DNA 双链氢键断裂；退火 50–65 °C，引物与互补序列结合；延伸 72 °C，Taq 聚合酶添加核苷酸。

The number of DNA copies doubles after each cycle: after n cycles, the number is 2ⁿ times the original.

每个循环后 DNA 拷贝数加倍：经过 n 个循环，拷贝数为原来的 2ⁿ 倍。

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9. Genetic Engineering: Transformation | 基因工程：转化

Transformation is the uptake of foreign DNA by a bacterial cell. Bacteria are often made competent by treatment with calcium chloride and heat shock.

转化是指细菌细胞摄取外源 DNA 的过程。常用氯化钙处理并热激使细菌变为感受态。

• Plasmids are used as vectors; they contain a multiple cloning site, a selectable marker (e.g. antibiotic resistance gene), and often a reporter gene.

  常用质粒作为载体；质粒含有多克隆位点、选择标记（如抗生素抗性基因），有时还包含报告基因。

• To identify transformed cells, plate bacteria on agar containing the antibiotic; only cells carrying the plasmid survive.

  为了筛选转化细胞，将细菌涂布在含抗生素的琼脂上；只有携带质粒的细胞能存活。

• Insertional inactivation: the foreign DNA inserts into the lacZ gene, so successful clones do not hydrolyse X-gal and remain white; unsuccessful clones produce blue colonies.

  插入失活：外源 DNA 插入 lacZ 基因后，成功克隆不能水解 X-gal 而呈白色；未插入的克隆呈蓝色。

Electroporation is an alternative method: a brief electric pulse creates temporary pores in the cell membrane, allowing DNA to enter.

电穿孔法是另一种方法：短暂电脉冲在细胞膜上形成临时小孔，使 DNA 进入。

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10. Chromatography | 层析法

Chromatography separates mixtures of molecules based on differences in their movement through a stationary phase under the influence of a mobile phase.

层析法利用混合物中各组分在固定相和流动相中运动速度不同进行分离。

• Paper chromatography uses paper as the stationary phase and a solvent as the mobile phase.

  纸层析以滤纸为固定相，溶剂为流动相。

• Thin-layer chromatography (TLC) uses a glass or aluminium plate coated with silica gel or cellulose.

  薄层层析（TLC）使用涂有硅胶或纤维素的玻璃板或铝板。

• The retention factor (Rf) is the ratio of the distance moved by the solute to the distance moved by the solvent front.

  比移值（Rf）是溶质移动距离与溶剂前沿移动距离之比。

Rf = Distance moved by solute ÷ Distance moved by solvent front

Rf = 溶质移动距离 ÷ 溶剂前沿移动距离

Rf values can be compared with known standards to identify substances.

通过与已知标准品比较 Rf 值可鉴定物质。

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11. Plant Tissue Culture | 植物组织培养

Plant tissue culture uses explants (small pieces of plant tissue) to grow whole plants on sterile nutrient media containing plant growth regulators.

植物组织培养利用外植体（小块植物组织）在含植物生长调节剂的无菌培养基上培育成完整植株。

• Medium contains a carbon source (sucrose), mineral salts, vitamins, and hormones such as auxins and cytokinins.

  培养基含有碳源（蔗糖）、无机盐、维生素以及生长素和细胞分裂素等激素。

• High auxin:cytokinin ratio promotes root formation; low ratio promotes shoot formation.

  高比例生长素/细胞分裂素促进生根；低比例促进出芽。

• Explants are surface-sterilised with ethanol or sodium hypochlorite to remove contaminating microbes.

  外植体需用乙醇或次氯酸钠进行表面消毒，以去除污染微生物。

Micropropagation produces genetically identical plants rapidly and allows the production of disease-free plants.

微繁殖能快速产生遗传上相同的植株，并能培育无病植物。

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12. Experimental Design and Controls | 实验设计与对照

Biotechnology experiments require careful controls to ensure valid conclusions. Common controls include sterile water instead of culture, known positive samples, and negative samples without the target substance.

生物技术实验需要精心设置对照以确保结论有效。常见对照包括用无菌水代替培养液、已知阳性样品以及不含目标物质的阴性样品。

• In ELISA, a positive control (known antigen) and a negative control (no antigen) must be included on each plate.

  在 ELISA 中，每块板必须包含阳性对照（已知抗原）和阴性对照（无抗原）。

• In PCR, a negative control without template DNA is used to check for contamination.

  在 PCR 中，使用不含模板 DNA 的阴性对照以检查污染。

• Repeat samples should be taken to calculate means and error bars; statistical tests can then determine significance.

  应设置重复样品以计算平均值和误差棒；随后用统计检验判断显著性。

A common question asks: “Why is a control plate with sterile water used?” Answer: To confirm that no contamination occurred during the procedure and that any colonies observed come from the original sample.

常见问题：”为什么使用无菌水对照平板？”答：用于确认操作过程中没有污染，并且观察到的菌落确实来自原始样品。

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