Essential Lab Techniques & Safety in Biology | 生物考点：基础实验操作与注意事项

📚 Essential Lab Techniques & Safety in Biology | 生物考点：基础实验操作与注意事项

In A-Level Biology, practical skills are formally assessed, not just in coursework but in written examinations that test your understanding of experimental design, instrumentation, and safety. Examiners consistently reward candidates who demonstrate precision, logical sequencing, and an awareness of potential errors. This article consolidates the essential laboratory operations and their precautions that you must master.

在 A-Level 生物考试中，实验技能不仅是课程作业的一部分，更是笔试中考察实验设计、仪器操作和安全意识的重要内容。考官始终青睐那些展现出精确操作、逻辑严谨且对潜在误差有清晰认知的考生。本文系统整理了必须掌握的基础实验操作及其注意事项。

1. General Laboratory Safety Rules | 实验室通用安全规则

Before any practical work, you must wear a lab coat, safety goggles, and (when handling hazardous chemicals or stains) disposable gloves. Long hair must be tied back, and open-toed shoes are not permitted. Never eat, drink, or apply cosmetics in the laboratory, as chemicals can be transferred to your mouth or eyes.

进行任何实验操作前，必须穿戴实验服、护目镜，并在接触危险化学品或染液时佩戴一次性手套。长发必须扎起，禁止穿露趾鞋。实验室中严禁进食、饮水或化妆，以免化学品经手接触口眼造成伤害。

Know the location of safety equipment: eye wash stations, fire blankets, and first-aid kits. If a chemical spills on your skin, rinse immediately with copious water and inform your supervisor. Broken glass must never be picked up with bare hands — use a dustpan and brush.

务必知晓安全设备的位置：洗眼器、灭火毯和急救箱。若化学品溅到皮肤，立即用大量清水冲洗并报告老师。碎玻璃严禁徒手捡拾，应使用簸箕和毛刷清理。

2. Using a Microscope | 显微镜的使用

Carry the microscope with one hand under the base and the other gripping the arm. Place it on a flat, stable bench, away from the edge. Begin focusing using the low-power objective lens, and always watch the objective from the side when racking it down towards the slide to avoid crushing the specimen.

搬运显微镜时，一手托住镜座，一手握住镜臂。将显微镜置于平稳的台面上，远离桌缘。对焦时应先使用低倍物镜，并在物镜下降靠近玻片时从侧面注视，防止压碎标本。

Use the coarse adjustment knob to bring the specimen into rough focus, then fine-tune with the fine adjustment knob. Always focus by moving the objective away from the slide, never towards it while looking through the eyepiece. Calculate magnification using:

使用粗准焦螺旋粗略对焦，再用细准焦螺旋微调。对焦时应始终使物镜远离玻片，切勿在目镜中观察的同时向下转动旋钮。放大倍数计算公式为：

Total magnification = Eyepiece magnification × Objective lens magnification

总放大倍数 = 目镜放大倍数 × 物镜放大倍数

Clean lenses only with special lens paper — never use tissue or cloth, as they scratch the coating. When finished, rotate to the lowest objective, lower the stage, and cover the microscope.

镜头只能用专用擦镜纸清洁，切勿使用纸巾或布，以免划伤镀膜。使用完毕后，将物镜转至最低倍率，载物台降至最低，并罩上防尘罩。

3. Preparing Temporary Mounts (Wet Mounts) | 临时装片的制备

A temporary mount is essential for observing living specimens such as onion epidermis, cheek cells, or aquatic organisms. Place the specimen in a drop of water (or stain) on a clean slide, then lower a coverslip at a 45° angle using a mounted needle. This technique excludes air bubbles, which would obstruct viewing and distort measurements.

临时装片用于观察活体材料，如洋葱表皮细胞、口腔上皮细胞或水生生物。在洁净载玻片上滴一滴水（或染液），放入标本，然后用解剖针以45°角缓慢放下盖玻片。该操作可避免产生气泡，气泡会阻碍观察并干扰测量。

If the specimen needs staining, add a drop of iodine solution (for starch-containing tissues), methylene blue (for animal cells), or methylene green. Excess stain is removed by touching a piece of filter paper to the edge of the coverslip — this draws the stain through by capillary action.

若需染色，可加入碘液（用于含淀粉组织）、亚甲基蓝（用于动物细胞）等。多余染液可用滤纸接触盖玻片边缘吸走，利用毛细作用使染液流经标本。

For quantitative work, remember the field of view diameter at low power (e.g., 4.5 mm) and calibrate the eyepiece graticule with a stage micrometer. To estimate cell length: divide the field diameter by the number of cells spanning it.

进行定量观察时，需记录低倍镜下的视野直径（如4.5 mm），并使用载物台测微尺校准目镜测微尺。估算细胞长度时，可将视野直径除以横跨该视野的细胞数量。

4. Accurate Pipetting and Measurement | 精确移液与测量

When using a graduated pipette, always read the liquid level at the bottom of the meniscus, with your eye at the same height as the meniscus to avoid parallax error. Use a pipette filler or pipette controller — never mouth-pipette dangerous chemicals. For volumes below 1 cm³, use a micropipette with disposable tips.

使用刻度吸管时，应读取弯月面最低点对应的刻度，视线须与弯月面保持水平，以避免视差误差。必须使用吸耳球或移液控制器，严禁用口吸取危险化学品。移取小于1 cm³的液体时，应使用带一次性枪头的微量移液器。

When using a measuring cylinder, choose the smallest cylinder that can hold your required volume for greatest accuracy. The uncertainty of a 10 cm³ measuring cylinder is typically ±0.2 cm³, whereas a 1 cm³ pipette has an uncertainty of only ±0.01 cm³. For maximum precision, select the most accurate instrument appropriate to the task.

使用量筒时，应选择能容纳所需体积的最小规格以提高精度。10 cm³量筒的典型不确定度为±0.2 cm³，而1 cm³移液管的不确定度仅为±0.01 cm³。为了最高精度，请根据任务选择最合适的仪器。

Balance the balance: always zero the top-pan balance before weighing.

天平校准：称量前务必使电子天平归零。
Weigh solids in a weighing boat or on filter paper to avoid contamination.

固体应在称量舟或滤纸上称量，避免污染。
Add solutions gradually, mixing thoroughly between additions.

溶液应逐滴加入，每次加入后充分混匀。

5. Preparing Dilution Series | 稀释系列的配制

Serial dilution is a fundamental technique for preparing a range of concentrations, e.g., for a calibration curve in colorimetry or for counting viable bacteria. To prepare a ten-fold dilution series, add 1 cm³ of stock solution to 9 cm³ of distilled water and mix thoroughly. Then transfer 1 cm³ of this solution to a second tube containing 9 cm³ of water, and repeat.

连续稀释（系列稀释）是配制浓度梯度的基本技术，例如用于比色法的标准曲线或细菌活菌计数。制备十倍稀释系列时，取1 cm³原液加入9 cm³蒸馏水中并充分混匀，再从该管中取1 cm³加入另一支含9 cm³水的试管中，如此重复。

Each step produces a 10⁻¹ (one-tenth) dilution of the previous solution. After n transfers, the concentration factor is 10⁻ⁿ. Always use a fresh pipette tip for each step to prevent carry-over of concentrated solution, and vortex or invert each tube for at least 5 seconds before sampling.

每一步都产生前一溶液10⁻¹（十分之一）的稀释度。经过n次转移后，浓度因子为10⁻ⁿ。每一步都必须更换新的枪头，防止浓溶液残留被带入下一管；取样前应涡旋或颠倒混匀至少5秒。

C₁V₁ = C₂V₂

C₁V₁ = C₂V₂（稀释公式）

Use the formula above when preparing a single dilution to a specific concentration. For example, to make 50 cm³ of 0.2 mol dm⁻³ glucose from 1.0 mol dm⁻³ stock: V₁ = (0.2 × 50) ÷ 1.0 = 10 cm³, so measure 10 cm³ stock and add 40 cm³ water.

配制特定浓度的单次稀释时，使用上式。例如：用1.0 mol dm⁻³葡萄糖原液配制50 cm³ 0.2 mol dm⁻³溶液：V₁ =（0.2 × 50）÷ 1.0 = 10 cm³，即取10 cm³原液加水至50 cm³。

6. Using a Colorimeter | 比色计的使用

A colorimeter measures the absorbance or percentage transmission of light through a coloured solution. It is used to determine concentrations of reducing sugars (using Benedict’s reagent), protein (Biuret test), or enzyme activity with artificial substrates.

比色计用于测量有色溶液对光的吸光度或透光率。常用于测定还原糖（本尼迪特试剂）、蛋白质（双缩脲试剂）的浓度，或通过人工底物测定酶活性。

First, select the appropriate filter (wavelength). A green filter (approximately 520-550 nm) is used for blue/green-coloured solutions, while a red filter (620-700 nm) suits blue solutions. The chosen wavelength should be the one at which the solution absorbs light most strongly for maximum sensitivity.

首先选择合适波长的滤光片。绿色滤光片（约520-550 nm）适用于蓝绿色溶液，红色滤光片（620-700 nm）适用于蓝色溶液。所选波长应为溶液吸收最强的波长，以获得最大灵敏度。

Calibrate the colorimeter using a blank cuvette containing only distilled water (or the solvent used in the test). Set the absorbance to zero or transmittance to 100%. Wipe the outside of each cuvette with lens tissue, and insert it with the same orientation each time to avoid variability from scratches.

使用仅含蒸馏水（或实验所用溶剂）的空白比色皿校准仪器，将吸光度调零或将透光率设为100%。每次测量前用擦镜纸擦拭比色皿外壁，并以同一方向插入样品槽，避免划痕造成误差。

Remember Beer-Lambert’s law: absorbance (A) is directly proportional to concentration (c) at low concentrations, where l is the path length and ε is the molar absorptivity:

牢记比尔-朗伯定律：在低浓度下，吸光度（A）与浓度（c）成正比，其中l为光程长度，ε为摩尔吸光系数：

A = εcl

7. Paper Chromatography | 纸层析法

Chromatography separates components of a mixture based on their differential solubility in the mobile phase and their affinity for the stationary phase. In paper chromatography, the stationary phase is the water absorbed in cellulose fibres, and the mobile phase is the solvent (e.g., ethanol:ammonia for leaf pigments).

层析法根据混合物中各组分在流动相中的溶解度差异及其对固定相的亲和力差异进行分离。纸层析中，固定相是纤维素纤维中吸收的水分，流动相是溶剂（如分离叶片色素的乙醇∶氨水）。

Draw the origin line in pencil — ink will dissolve and interfere with separation. Apply the sample as a small concentrated spot using a capillary tube, and allow it to dry fully between applications to create a concentrated start. Suspend the paper in the chromatography tank so that the origin line is above the solvent surface.

原点线必须用铅笔画——墨水会溶解并干扰分离。用毛细管将样品点成小而集中的斑点，每次点样后待其完全干燥再重复点样以浓缩样品。将滤纸悬挂于层析缸中，确保原点线在溶剂液面之上。

Keep the tank covered to maintain solvent saturation. When the solvent front is near the top of the paper, remove the paper and mark the solvent front immediately with pencil before it dries. Calculate the Rf value for each spot:

层析缸必须加盖以保持溶剂蒸气饱和。当溶剂前沿接近滤纸顶端时取出滤纸，并立即用铅笔标记溶剂前沿（防止干燥后边界不清）。计算各斑点的Rf值：

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

8. Biological Staining Techniques | 生物染色技术

Most biological specimens are transparent under the light microscope; stains increase contrast by binding to specific cellular structures. The choice of stain depends on the structure you wish to highlight.

大多数生物标本在光学显微镜下呈透明状；染色剂通过与特定细胞结构结合来增强对比度。染色剂的选择取决于你想要凸显的结构。

Iodine solution: stains starch granules blue-black; used for onion cells and potato sections.

碘液：将淀粉粒染成蓝黑色，用于洋葱细胞和马铃薯切片。
Methylene blue: stains nuclei blue in animal cells (e.g., cheek epithelial cells).

亚甲基蓝：将动物细胞（如口腔上皮细胞）的细胞核染成蓝色。
Acetic orcein / aceto-carmine: stain chromosomes dark red/pink in root tip squash preparations.

醋酸地衣红 / 醋酸卡红：将根尖压片中的染色体染成深红色/粉红色。
Sudan III / Sudan black: stains lipid droplets orange/black in plant or animal tissue.

苏丹III / 苏丹黑：将动植物组织中的脂滴染成橙色/黑色。

For the root tip squash, warm the root tip in 1 mol dm⁻³ hydrochloric acid to soften it and separate cells, then rinse thoroughly before staining — residual acid will distort chromosome morphology. Apply gentle pressure to the coverslip using a mounted needle (covered with filter paper) to spread the cells into a single layer.

制作根尖压片时，先用1 mol dm⁻³盐酸加热处理根尖以软化和分离细胞，染色前必须彻底冲洗——残留酸会破坏染色体形态。用解剖针隔着滤纸对盖玻片轻轻施压，使细胞分散成单层。

9. Measuring Enzyme Activity | 酶活性的测定

Enzyme assays are among the most common practical assessments. The rate of reaction is measured by monitoring the disappearance of substrate or the appearance of product over time. Methods include measuring gas production (e.g., catalase + hydrogen peroxide → oxygen), colour change (e.g., amylase + starch → reducing sugar), or change in pH.

酶活性测定是最常见的实验考查内容之一。通过监测底物的消耗或产物的生成随时间的变化来计算反应速率。常用方法包括测量气体产量（如过氧化氢酶 + 过氧化氢 → 氧气）、颜色变化（如淀粉酶 + 淀粉 → 还原糖）或pH变化。

Control the temperature precisely using a water bath set to the desired temperature (±0.5°C), and pre-incubate the enzyme and substrate separately for 5 minutes before mixing — this ensures both reach thermal equilibrium. Use a buffer to maintain a constant pH, as changes in H⁺ concentration alter enzyme tertiary structure.

使用水浴将温度精确控制在目标值（±0.5°C），酶和底物须分别预保温5分钟后再混合，以确保两者达到热平衡。使用缓冲液维持恒定pH，因为H⁺浓度变化会改变酶的三级结构。

Stop the reaction at timed intervals by adding a “stopping reagent” (e.g., hydrochloric acid or boiling). For example, in the amylase-starch assay, remove a 1 cm³ sample at each time point and add it to iodine in a spotting tile — the blue-black colour indicates remaining starch.

在设定时间间隔加入”终止剂”（如盐酸或沸水浴）终止反应。例如，在淀粉酶-淀粉实验中，每个时间点取出1 cm³样品加入白瓷板上的碘液中——出现蓝黑色表明仍有淀粉存在。

Always run a negative control (without enzyme or with boiled enzyme) and a positive control (known active enzyme). Use at least three replicates at each condition and calculate the mean rate; discard anomalous results only if you can identify a procedural error.

务必设置阴性对照（不含酶或使用煮沸失活的酶）和阳性对照（已知活性正常的酶）。每个条件下至少设置三组重复并计算平均速率；只有能确认操作失误时才可舍弃异常数据。

10. Centrifugation | 离心技术

Centrifugation separates cell organelles or cellular components by density. This technique is central to practicals such as extracting chloroplasts for the Hill reaction, or separating DNA from cell debris.

离心技术根据密度差异分离细胞器或细胞组分。该技术广泛应用于叶绿体提取（希尔反应）或从细胞碎片中分离DNA等实验。

Balance the centrifuge tubes carefully: each tube must be matched by another of equal mass placed directly opposite in the rotor. An unbalanced rotor causes severe vibration, can damage the instrument, and risks personal injury. Always close the lid before starting, and never open the centrifuge until it has completely stopped.

离心管必须对称配平：每支试管需在转子对面放置质量相等的配对管。转子不平衡会引起剧烈振动，损坏仪器并存在人身伤害风险。启动前务必盖好盖子，离心机完全停止前严禁打开。

After centrifugation, the denser material forms a pellet at the bottom; the supernatant is the liquid above. To collect the pellet, carefully decant or pipette off the supernatant without disturbing the pellet. To wash a pellet, resuspend it in fresh buffer and centrifuge again.

离心结束后，密度较大的物质在管底形成沉淀（pellet），上清液（supernatant）位于上层。收集沉淀时应小心倒出或用移液器吸走上清液，避免搅动沉淀。若需洗涤沉淀，可用新鲜缓冲液重悬后再离心一次。

Record centrifugation conditions in your results: relative centrifugal force (RCF) in × g, temperature, and duration. Chilled centrifuges (4°C) are essential for enzyme or organelle extraction to prevent protein denaturation.

记录离心条件：相对离心力（RCF，单位× g）、温度和时长。提取酶或细胞器时须使用4°C低温离心机，以防止蛋白质变性。

11. Aseptic Technique | 无菌操作技术

Aseptic technique is vital in microbiology practicals — culturing bacteria or fungi, or investigating the effect of antibiotics. The goal is to exclude contaminating microorganisms from your culture and to prevent laboratory-acquired infections.

无菌操作技术在微生物学实验中至关重要——培养细菌或真菌、研究抗生素效果等。其目标是防止杂菌污染培养物，同时避免实验室获得性感染。

Sterilise equipment before use. Autoclaving at 121°C, 15 psi for 15 minutes kills all microorganisms, including endospores. Work near a Bunsen burner flame — the rising convection current carries airborne contaminants away from your sterile area. Flame the inoculating loop to red heat before and after every transfer.

使用前必须灭菌。高压蒸汽灭菌（121°C、15 psi、15分钟）可杀死包括内生孢子的所有微生物。在酒精灯火焰附近操作——上升的热气流将空气中的污染物带离无菌区域。接种环每次使用前后均须灼烧至红热。

Open petri dishes only at an angle, never fully, and replace the lid immediately.

培养皿只能斜开一条缝，切勿完全打开，用后立即盖回。
Flame the neck of glass bottles and test tubes before and after removing samples.

玻璃瓶和试管口在取样前后均须过火灭菌。
Use sterile pipettes and tips, and never touch the sterile surfaces.

使用无菌移液管和枪头，切勿触碰无菌表面。
Label plates on the bottom (agar side), not the lid, to prevent misidentification if lids are swapped.

在培养皿底面（含培养基的一侧）标注标签，而非盖子上，防止盖子互换导致混淆。

Incubate plates upside down (inverted) to prevent condensation droplets from dripping onto the agar surface and spreading colonies. For safety, school cultures are typically incubated at 25°C, not 37°C, to reduce the growth of harmful human pathogens.

培养皿应倒置培养，防止冷凝水滴落至培养基表面导致菌落蔓延。出于安全考虑，学校实验通常在25°C而非37°C培养，以减少人体病原菌的生长风险。

12. Common Errors and How to Avoid Them | 常见误差及避免方法

Examiners penalise poor experimental technique. Understanding the types of error and their remedies demonstrates higher-order thinking. Random errors arise from unpredictable fluctuations — parallax in reading volumes, timing inconsistencies, or slight temperature drift. They reduce precision but can be minimised by taking repeat measurements and calculating the mean.

考官会因实验操作不当而扣分。理解误差类型及其纠正方法体现了高阶思维。随机误差来自不可预测的波动——读数视差、计时不一致或细微温度漂移。随机误差降低精密度，但可通过重复测量取平均值来减小。

Systematic errors affect accuracy in one direction, such as a miscalibrated balance that always reads 0.05 g high, or using a thermometer with an incorrect calibration. These cannot be corrected by repeats; you must calibrate instruments and validate them against known standards.

系统误差使结果单向偏离真实值，例如未校准的天平始终偏大0.05 g，或温度计刻度不准。此类误差无法通过重复测量消除，必须校准仪器并用已知标准验证。

Common error 常见误差	Cause 成因	Remedy 纠正方法
Parallax error 视差	Eye not level with meniscus 视线未与弯月面水平	Read at eye level 视线平视读数
Contamination 污染	Reusing tips or touching surfaces 枪头重复使用或接触表面	Fresh tip per transfer; sterile technique 每步换新枪头；无菌操作
Inaccurate timing 计时不准	Starting/stopping at wrong moment 起止时间不当	Use a stopwatch; add reagents with a consistent trigger 使用秒表；动作统一
Air bubbles in mount 装片气泡	Coverslip dropped flat 盖玻片水平放下	Lower at 45° angle 以45°角缓慢放下

To improve reliability, repeat each measurement at least three times and calculate the mean. If one result differs markedly from the others, record it but do not discard it without justification. Reliability is improved by wider sampling and longer observation, while validity is improved by controlling all variables except the independent one.

提高可靠性，每项测量至少重复三次并计算平均值。若某一结果明显偏离其他数据，应予以记录，但不可无理由舍去。增大样本量和延长观察时间可提高可靠性；控制除自变量以外的所有变量可提高有效性。

Finally, always record results with appropriate units and significant figures consistent with the precision of your instruments. Write down any anomalous observations — they often reveal useful insights about the biological system or your technique.

最后，记录结果时须包含正确的单位，有效数字位数应与仪器精度一致。记录一切异常观察结果——这些异常往往能揭示生物系统或操作技巧中的重要信息。

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Essential Lab Techniques & Safety in Biology | 生物考点：基础实验操作与注意事项