计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
细菌活力/毒性检测试剂盒包含两种荧光染料，NucGreen 是绿色核酸染料，可染色活细菌和死细菌；EthD-III 是红色核酸染料，仅染色细胞膜受损的死细菌。将 NucGreen 和 EthD-III 适当混用，具有完整细胞膜的细菌呈现绿色，而具有受损细胞膜的细菌在不同通道下可分别呈现绿色和红色。 细菌活力的常见标准是在合适的营养培养基中繁殖的能力，称为生长测定。本试剂盒通常与在液体或固体培养基中的生长测定结果有着很好的一致性。然而，在某些条件下，膜损伤的细菌可能会在营养培养基中恢复并繁殖，而这种细菌在本测定中会被认定死菌。相反，一些具有完整膜的细菌可能无法在营养培养基中繁殖，但在本测定中会被认认定活菌。因此，若本试剂盒检测结果和细菌生长测定之间有较大的差异，应考虑上述可能。
组分：
产品参数：
NucGreen: Ex/Em = 503/530 nm (结合 DNA)；EthD-III: Ex/Em = 530/620 nm (结合 DNA)。
注意事项：
1. 使用前请将产品瞬时离心至管底，再进行后续实验。
2. 若使用孔板检测，可静置 10 min 后留少量菌液成像，能有效降低背景。
3. 为更接近真实结果，Merge图片时建议保持红色荧光与绿色荧光亮度一致。
4. 荧光染料均存在淬灭问题，实验操作时请尽量注意避光，以减缓荧光淬灭。
5. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用方法：
一. 活、死细菌样品对照制备（可选）
1. 在液体培养基中培养4 mL的细菌至晚期对数期。
2. 在EP管中准备两份1 mL的细菌液，并在5,000~10,000 g条件下离心10~15 min。
3. 去除上清液，在其中一支EP管中加入0.3 mL的0.85% NaCl重悬细菌，在另一管中加入1 mL的0.85% NaCl重悬细菌。
4. 在含有0.3 mL的0.85% NaCl的管中加入0.7 mL异丙醇，充分混合（最终浓度为70%的异丙醇）用以制备死细菌样品。
5. 将两种样品在室温下孵育1 h，每15 min混合一次。
6. 两种样品在5,000~10,000 g条件下离心10~15 min。
7. 去除上清，在两种样品中加入1 mL的0.85% NaCl重悬细菌，并如步骤6再次离心。
8. 使用分光光度计测定两种菌悬液在670 nm处的吸光值（OD670）。
9. 将两种菌悬液（活的和死亡的）密度调整到108 个细菌/ mL（OD670 ≈ 0.3），然后用0.85% 的NaCl以1：100稀释，使最终 密度为106 个细菌/ mL。
10. 如下表所示混合两种菌悬液以获得所需的活细胞：死细胞比率。
表 1. 活、死菌悬液按一定体积混合以达到所需的活细胞、死细胞比例
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二. 荧光显微镜观察的染色方法
1. 将1体积的A组分NucGreen和2体积的B组分EthD-III在微量离心管中混合，充分混合后加入8体积的0.85% NaCl溶液以得 到100×染料溶液。
2. 每100 μL菌悬液，加1 μL的100×染料溶液。
3. 充分混合，室温下在黑暗中孵育15 min。
4. 取5 μL染色后的菌悬液滴在带有18 mm方形盖玻片的载玻片上。
5. 在荧光显微镜下观察。活细菌和死细菌的荧光可以在任何标准的FITC长效过滤器下同时观察到。或者，活的（绿色荧 光）和死的（红色荧光）细菌可以分别用FITC和Cy3（或Texas Red）通道观察。
注意： (1) 在对细菌染色之前，必须注意除去生长介质的残留。核酸和其他培养基组分可以以某种方式结合NucGreen和EthD-III染 料，导致不可接受的染色变化。简单的洗涤步骤通常足以从细菌悬浮液中除去干扰介质组分。不建议使用磷酸盐缓冲液， 因为它们会降低染色效率。 (2) 在开始正式实验前应调节染料浓度来使NucGreen标记活细菌、使EthD-III标记死细菌进行区分。最佳浓度可能因细菌菌 株的不同而异。一般最好使用能够提供充足信号的最低染料浓度。以上条件针对大肠杆菌活/死细胞染色进行了优化。
三. 流式细胞仪的染色方法 实验开始前，请阅读荧光显微镜染色步骤下的注意事项。
1. 根据表1，在EP管中加入11种不同比例的活细菌和死细菌。11种样品中每种的体积1 mL。
2. 将12 μL的A组分NucGreen与24 μL的B组分EthD-III在微量离心管中混合。11个样品中的每个加入3 μL的混合染料，通过 上下吹打几次彻底混合。（注意：需要准备另外的对照细菌样品用于单独的NucGreen和单独的EthD-III染色）
3. 室温下在黑暗中孵育15 min。
4. 使用流式细胞仪分析每个样品，使用 FITC 通道检测 NucGreen 阳性细胞的，使用 PI 或 PE 通道检测 EthD-III 阳性细胞。
应用范围：
死、活细菌染色
The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, the bacteria with intact cell membrane appear green, while the bacteria with damaged cell membrane can appear green and red under different channels, respectively. A common criterion for bacterial viability is the ability to propagate in a suitable nutrient medium, known as a growth assay. This kit is generally in good agreement with the growth assay results in liquid or solid medium. However, under certain conditions, membrane damaged bacteria may recover and propagate in nutrient medium, and such bacteria will be identified as dead bacteria in this assay. On the contrary, some bacteria with intact membranes may not be able to propagate in nutrient medium, but will be recognized as viable bacteria in this assay. Therefore, if there is a large difference between the test results of this kit and the bacterial growth assay, the above possibilities should be considered.
Component：
Product parameters：
NucGreen: Ex/Em = 503/530 nm (结合 DNA)；EthD-III: Ex/Em = 530/620 nm (结合 DNA)。
Usage:
1 Preparation of control samples for live and dead bacteria (optional)
1. Cultivate 4 mL of bacteria in liquid medium until late logarithmic phase.
2. Prepare two 1 mL bacterial solutions in an EP tube and centrifuge for 10-15 minutes under 5000-10000 g conditions.
3. Remove the supernatant and add 0.3 mL of 0.85% NaCl resuspended bacteria to one of the EP tubes, and 1 mL of 0.85% NaCl resuspended bacteria to the other tube.
4. Add 0.7 mL of isopropanol to a tube containing 0.3 mL of 0.85% NaCl, and mix thoroughly (with a final concentration of 70% isopropanol) to prepare a dead bacterial sample.
5. Incubate the two samples at room temperature for 1 hour and mix every 15 minutes.
6. Centrifuge the two samples at 5000-10000 g for 10-15 minutes.
7. Remove the supernatant, add 1 mL of 0.85% NaCl to resuspend the bacteria in both samples, and centrifuge again as in step 6.
8. Use a spectrophotometer to measure the absorbance values (OD670) of two bacterial suspensions at 670 nm.
9. Adjust the density of the two bacterial suspensions (live and dead) to 108 bacteria/mL (OD670 ≈ 0.3), and then dilute with 0.85% NaCl at 1:100 to achieve a final density of 106 bacteria/mL.
10. Mix two bacterial suspensions as shown in the table below to obtain the required live cell ratio: dead cell ratio.
Table 1 Mix live and dead bacterial suspensions by a certain volume to achieve the required ratio of live and dead cells
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II Staining methods for fluorescence microscopy observation
1. Mix 1 volume of component A, NucGreen, and 2 volumes of component B, EthD-III, in a microcentrifuge tube. After thorough mixing, add 8 volumes of 0.85% NaCl solution to obtain a 100 x dye solution.
2. Every 100 μ L bacterial suspension, add 1 μ 100 x dye solution of L.
3. Mix thoroughly and incubate at room temperature in the dark for 15 minutes.
4. Take 5 μ The bacterial suspension after L staining was dropped onto a glass slide with an 18 mm square cover glass.
5. Observe under a fluorescence microscope. The fluorescence of live and dead bacteria can be observed simultaneously under any standard FITC long-acting filter. Alternatively, live (green fluorescent) and dead (red fluorescent) bacteria can be observed using FITC and Cy3 (or Texas Red) channels, respectively.
Attention: (1) Before staining bacteria, attention must be paid to removing residues of growth media. Nucleic acid and other media components can bind to NucGreen and EthD-III dyes in some way, resulting in unacceptable staining changes. A simple washing step is usually sufficient to remove interfering media components from bacterial suspension. It is not recommended to use phosphate buffer solutions as they can reduce staining efficiency. (2) Before starting the formal experiment, the dye concentration should be adjusted to distinguish between NucGreen labeling live bacteria and EthD-III labeling dead bacteria. The optimal concentration may vary depending on the bacterial strain. It is generally best to use the lowest dye concentration that can provide sufficient signal. The above conditions have been optimized for staining live/dead cells of Escherichia coli.
III Before starting the staining method experiment of flow cytometry, please read the precautions under the fluorescence microscope staining steps.
According to Table 1, add 11 different proportions of live and dead bacteria to the EP tube. Each of the 11 samples has a volume of 1 mL.
2. Add 12 μ The A component of L, NucGreen, and 24 μ The B component EthD-III of L was mixed in a microcentrifuge tube. Add 3 to each of the 11 samples μ Mix the mixed dyes of L thoroughly by blowing them up and down several times. (Note: Additional control bacterial samples need to be prepared for separate NucGreen and EthD-III staining)
3. Incubate at room temperature in the dark for 15 minutes.
4. Analyze each sample using a flow cytometer, detect NucGreen positive cells using FITC channels, and detect EthD-III positive cells using PI or PE channels.
Matters needing attention：
1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments.
2. if the orifice plate is used for detection, a small amount of bacterial liquid can be left for imaging after standing for 10 min, which can effectively reduce the background.
3. in order to be closer to the real results, it is recommended to keep the brightness of red fluorescence consistent with that of green fluorescence in merge pictures.
4. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching.
5. for your safety and health, please wear experimental clothes and disposable gloves.
Scope of application：
Staining of dead and live bacteria
规格
英文名称
Bacterial viability / toxicity test kit
储存与运输
储存条件
避光,-20°C储存
运输条件
超低温运输
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 H2402452 |
分析证书 | B598379 |
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溶液计算器
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