计算溶液所需的质量、体积或浓度。
细胞培养污染检测试剂盒
有货
为什么选择此级别
BioReagent,无菌过滤 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
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
是否无菌：是
细胞培养污染检测试剂盒提供一种在细胞培养过程中简单有效的进行微生物污染检测的方法。细胞培养污染检测试剂盒不仅用于检测是否存在污染物，同时还可以鉴别污染物类型，包括酵母（和其他真菌）、革兰氏阳性菌和革兰氏阴性菌。
使用本试剂盒时，需要将样品进行两个载玻片的染色：一个载玻片上用钙荧光白 M2R染色，它是一种可被紫外激发的、特异性结合真菌细胞壁的蓝色荧光染料。
另一个载玻片是用JJ98 绿色核酸染料和WGA-AF 594染色，JJ98 是一种绿色荧光核酸染料；WGA 是一种麦胚凝集素，可以结合 N-乙酰葡萄糖胺和 N-乙酰神经氨酸残基。由于 N-乙酰葡萄糖胺在革兰氏阳性菌中的丰度和可获得性通常大于革兰氏阴性菌，所以 WGA 的荧光偶联物可作为革兰氏阳性菌的探针。因此，第二个载玻片可鉴别出革兰氏阳性和阴性菌。革兰氏阳性菌可被同时染上红色和绿色荧光，革兰氏阴性菌只呈现绿色荧光。
本试剂盒具有以下特点：
1. 可在一小时内通过荧光染色判断培养细胞是否存在微生物污染。
2. 可直接鉴定出所染菌的大致种类，从而为下一步制定解决方案提供依据。
3. 染色液为溶液形式，无需溶解，可直接进行工作液的配制。
4. 染色清晰，荧光明亮，灵敏度高，结果更加准确。
产品组成
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使用说明
一、试剂和工作液的准备：
1. BSA-生理盐水溶液：将 250 mg BSA 和 0.88g NaCl 溶解于 100mL 蒸馏水中，过滤除菌，配制成含有 0.25% BSA 和 0.15 M NaCl 的 BSA-生理盐水溶液。（本试剂盒不提供，请自行配制）
2. WGA-AF 594工作液：2.5 μL WGA-AF 594+47.5 μL BSA-生理盐水溶液。
3. JJ98 绿色核酸染料工作液：0.5 μL JJ98 绿色核酸染料+9.5 μL 蒸馏水。
4. 钙荧光白 M2R工作液：0.5 μL 钙荧光白 M2R+9.5 μL 蒸馏水。
注：工作液可在室温下稳定数小时。
二、样品的准备：
1．取 5 mL细胞培养物样本，将其转移到 15 mL无菌离心管中。对于贴壁细胞，只吸取
培养基上清；对于悬浮细胞，需要将细胞和培养基一起收集。
2．1000×g 离心 15 分钟。
3．小心弃掉上清，用 200 μL BSA-生理盐水溶液重悬。
注：可先用 1 mL BSA-生理盐水溶液重悬进行洗涤，离心后再用 200 μL BSA-生理盐水溶液重悬，这样可洗掉培养基中核酸和其他成分，减少染料的非特异结合。
4．分别将 20 μL 样品滴加在两个用乙醇或甲醇预清洗的载玻片上，分别标记“B”和“Y”，“B”用于细菌检测，“Y”用于真菌检测。
注：建议用 PAP PAN 或其他免疫组化笔在载玻片上画圈，以限制液滴。
5．将滴加有样品的载玻片置于 37℃下干燥约 10 分钟。然后样品面朝上，快速过火焰三次，进行固定。
注：注意不要过热，每过一次火焰可用手背感受温度，控制在皮肤可忍受的温度。
6．将 10 μL 稀释后的钙荧光白 M2R工作液滴加于载玻片 Y 的干燥样品上，使其均匀覆盖。小心覆盖盖玻片，避免产生气泡，避光保存。
注：覆盖盖玻片后可用石蜡封边。
7．将 50 μL BSA-生理盐水溶液滴加于载玻片 B 的干燥样品上，使其均匀覆盖。静置 5分钟，小心吸弃液体。
8．将 50 μL 稀释后的WGA-AF 594工作液滴加于上述载玻片 B 的样品上，使其均匀覆盖。静置 5 分钟，小心吸弃液体。
9．将 10 μL 稀释后的 JJ98 绿色核酸染料工作液滴加于上述载玻片 B 的样品上，使其均匀覆盖。小心覆盖盖玻片，避免产生气泡，避光保存。
注：为减少绿色荧光背景，可先用 BSA-生理盐水溶液洗去工作液，然后再覆盖盖玻片。覆盖盖玻片后可用石蜡封边。
三、荧光显微镜观察：
1. 需用 400 倍或 1000 倍（需使用油镜）放大进行观察。
2. 为了观察真菌染色情况（载玻片 Y），可根据下表激发和发射波长选择合适通道进行蓝色荧光的显微镜观察。酵母菌可被染上明亮的蓝色荧光，呈圆形或椭圆形，常有出芽；丝状真菌也可呈现丝状蓝色荧光。相比之下，细菌几乎不被染色。
3. 为了观察细菌染色情况（载玻片 B），首先根据下表激发和发射波长选择合适通道进行绿色荧光的显微镜观察，革兰氏阳性菌和革兰氏阴性菌均会呈现明亮的绿色荧光。（形态不规则的绿色荧光染色物可能是灰尘或细胞碎片。）然后根据下表激发和发射波长选择合适通道进行红色荧光的显微镜观察，革兰氏阳性菌可呈现明亮的红色荧光，而革兰氏阴性菌几乎不被染色。
注：真菌也会染上绿色或红色荧光，但是很容易通过它们的形态和大小差异区分出来。
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注意事项
1. 管中液体体积较小，请于每次吸取前先瞬离，减少液体损失。
2. 为保持更好的检测效果，WGA-AF 594 建议 2.5 μL / 管分装后保存，所有染料工作液现用现配。
3. 荧光染料都存在淬灭的问题，建议在检测过程中注意避光，染色后尽量当天完成检测。
4. 为了您的安全和健康，操作时请穿着实验服并佩戴一次性手套。
The Cell Culture Contamination Detection Kit provides a simple and effective method for microbial contamination testing during cell culture. This kit not only detects the presence of contaminants but also identifies contaminant types, including yeast (and other fungi), Gram-positive bacteria and Gram-negative bacteria.
When using this kit, samples are stained on two separate slides: One slide is stained with Calcofluor White M2R, a UV-excitable blue fluorescent dye that specifically binds to fungal cell walls.
The other slide is stained with MycoLight Green JJ98 nucleic acid dye and WGA-iFluor 594. MycoLight Green JJ98 is a green fluorescent nucleic acid stain. WGA (Wheat Germ Agglutinin) binds to N-acetylglucosamine and N-acetylneuraminic acid residues. Since N-acetylglucosamine is generally more abundant and accessible in Gram-positive bacteria than in Gram-negative bacteria, fluorescent WGA conjugates serve as a probe for Gram-positive bacteria. Accordingly, the second slide differentiates Gram-positive from Gram-negative bacteria: Gram-positive bacteria exhibit both red and green fluorescence, while Gram-negative bacteria show only green fluorescence.
Key features of this kit:
1.Microbial contamination in cell cultures can be determined within one hour via fluorescent staining.
2.It enables direct preliminary classification of stained microbes to support subsequent troubleshooting strategies.
3.Dyes are supplied as ready-to-use solutions; no powder reconstitution is required for working solution preparation.
4.Bright, distinct fluorescent signals with high sensitivity for reliable and accurate results.
Components
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Instructions for Use
I. Preparation of Reagents and Working Solutions
1.BSA-saline solution: Dissolve 250 mg BSA and 0.88 g NaCl in 100 mL distilled water, then filter-sterilize to prepare BSA-saline solution containing 0.25% BSA and 0.15 M NaCl. (Not supplied in this kit; prepare separately.)
2.WGA-iFluor 594 working solution: Mix 2.5 μL WGA-iFluor 594 with 47.5 μL BSA-saline solution.
3.MycoLight Green JJ98 working solution: Mix 0.5 μL MycoLight Green JJ98 with 9.5 μL distilled water.
4.Calcofluor White M2R working solution: Mix 0.5 μL Calcofluor White M2R with 9.5 μL distilled water.
Note: Working solutions remain stable for several hours at room temperature.
II. Sample Preparation
1.Transfer 5 mL cell culture sample into a 15 mL sterile centrifuge tube. For adherent cells, collect only the culture supernatant; for suspension cells, collect cells together with the medium.
2.Centrifuge at 1000×g for 15 minutes.
3.Carefully discard the supernatant and resuspend the pellet in 200 μL BSA-saline solution.
Note: A washing step is optional: resuspend the pellet in 1 mL BSA-saline solution, centrifuge, then resuspend again in 200 μL BSA-saline solution. This removes nucleic acids and other medium components to reduce non-specific dye binding.
4.Pipette 20 μL sample onto two ethanol/methanol-precleaned slides, labeled “B” (for bacteria detection) and “Y” (for fungi detection), respectively.
Note: Use a PAP pen or other immunohistochemical pen to draw a circle on the slide to confine the droplet.
5.Air-dry the sample-loaded slides at 37 °C for approximately 10 min. Fix the sample by quickly passing the slide (sample side up) through a flame three times.
Note: Avoid overheating. Check the temperature with the back of your hand after each pass; keep it tolerable to skin.
6.Pipette 10 μL diluted Calcofluor White M2R working solution onto the dried sample on Slide Y to fully cover it. Carefully apply a coverslip without trapping air bubbles, and store in the dark.
Note: Paraffin sealing is optional after coverslip placement.
7.Pipette 50 μL BSA-saline solution onto the dried sample on Slide B to fully cover it. Incubate for 5 min, then carefully aspirate the liquid.
8.Pipette 50 μL diluted WGA-iFluor 594 working solution onto the sample on Slide B to fully cover it. Incubate for 5 min, then carefully aspirate the liquid.
9.Pipette 10 μL diluted MycoLight Green JJ98 working solution onto the sample on Slide B to fully cover it. Carefully apply a coverslip without trapping air bubbles, and store in the dark.
Note: To reduce green background fluorescence, rinse with BSA-saline solution before coverslip placement. Paraffin sealing is optional after coverslip placement.
III. Fluorescence Microscopy Observation
1.Observe at 400× magnification or 1000× magnification (oil immersion required).
2.For fungal staining (Slide Y): Select the appropriate channel according to the excitation/emission wavelengths listed in the spectral table to visualize blue fluorescence. Yeast cells show bright blue, round or oval fluorescence, often with budding structures; filamentous fungi display thread-like blue fluorescence. In contrast, bacteria show negligible staining.
3.For bacterial staining (Slide B): First select the corresponding channel to detect green fluorescence. Both Gram-positive and Gram-negative bacteria exhibit bright green fluorescence. (Irregular green fluorescent particles may be dust or cell debris.) Then switch to the appropriate channel for red fluorescence observation: Gram-positive bacteria produce strong red signals, while Gram-negative bacteria are barely stained.
Note: Fungi may also show green or red fluorescence, but they can be readily distinguished from bacteria by morphology and size.
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Precautions
1.The volume of liquid in each tube is small. Briefly centrifuge the tubes before pipetting each time to avoid reagent loss.
2.For optimal detection performance, aliquot WGA-iFluor 594 into 2.5 μL portions for storage. Prepare all dye working solutions freshly right before use.
3.Fluorescent dyes are susceptible to photobleaching. Protect samples from light during operation and complete microscopic observation on the same day after staining.
4.Wear a lab coat and disposable gloves throughout the procedure for personal safety and health protection.
规格
别名
细胞微生物污染检测试剂盒，细胞培养细菌真菌污染检测试剂盒
英文别名
Molecular Probes Cell Culture Contamination Detection Kit
规格或纯度
BioReagent, 无菌过滤
英文名称
Cell Culture Contamination Detection Kit
应用
Microbial Contamination Detection
样品类型
细胞
检测方法
荧光法
定量&半定量
半定量
检测仪器
荧光显微镜
检测波长
Calcofluor White M2R: Ex :365 nm; Em: 435 nm MycoLight Green JJ98: Ex :482 nm; Em: 512 nm WGA-iFluor 594: Ex : 588 nm; Em: 604 nm
敏感性
Light-sensitive
Emission(nm)
435nm, 512nm, 604nm
Excitation(nm)
365nm, 482nm, 588nm
Ex/Em(nm)
Ex:365nm;Em:435nm,Ex:482nm;Em:512nm,Ex:588nm;Em:604nm
储存与运输
储存条件
避光,-20°C储存
运输条件
超低温运输
稳定性与储存
Store at -20℃ long term (12 months). Store in the dark.
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 20T | 100T | 储存 |
| C1527959A | 钙荧光白 M2R | 液体 | 10 μL | 50 μL | -20°C.Store in the dark. |
| C1527959B | JJ98 绿色核酸染料 | 液体 | 10 μL | 50 μL | -20°C.Store in the dark. |
| C1527959C | WGA-AF 594 | 液体 | 50 μL | 250 μL | -20°C.Store in the dark. |
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
技术文档和文章
溶液计算器
常见问题
本产品应如何储存？
请于?20 °C、避光条件下保存。本品对光敏感，请保存在原始的避光或棕色容器中。
什么是「无菌过滤」？
「无菌过滤」表示该物料经过加工和检测，适用于特种工业加工。规格侧重于影响该应用性能的杂质——例如，电子应用的金属含量、电池应用的粒度、光学应用的折射率，或对水分敏感工艺的水分含量。
什么是「BioReagent」？
「BioReagent」表示该产品经过加工和检测，适用于生物和生化应用。规格可能包括内毒素限值、微生物控制、无菌性、低 DNase/RNase/蛋白酶活性，以及在相关情况下的生物活性验证。
本产品如何运输？
本产品采用保温箱配冰袋运输。收货后请立即拆包，并按上述储存条件保存。
如何获取批次专用COA？
每个订单都会随附分析证书（COA），您也可以从账户订单历史中下载。如需订购前的COA，请联系您的客户代表，或使用PDP上的"申请COA"按钮——我们将提供代表性批次的COA。
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