活细菌/死细菌双染试剂盒荧光探针

SYTO 9/PI Live/Dead Bacterial Double Stain Kit

活细菌/死细菌双染试剂盒

产品信息

试剂盒规格说明（以MX4234-40T为例，按照建议的试剂稀释倍数和单次测试体积来计算）：

◇ 荧光显微镜检测，1ml菌液加入3μl染料混合液（SYTO 1.5μl +PI 1.5μl），可做40次独立测试。

◇ 荧光酶标仪检测，2ml无菌水加入12μl染料混合液（SYTO 6.0μl +PI 6.0μl），制备成2×工作液。按照100μl染料混合液加入100μl菌液的比例使用，可做200次独立测试。

◇ 流式细胞仪检测，2ml菌液加入6μl染料混合液（SYTO 3.0μl +PI 3.0μl），可做20次独立测试。

产品描述

活细菌/死细菌双染试剂盒（SYTO 9/PI Live/Dead Bacterial Double Stain Kit）是一款方便且操作简单的试剂盒，利用SYTO 9绿色核酸染料和碘化丙啶（PI）红色荧光核酸染料来进行细菌活力的检测，适用于大量的细菌种属，包括蜡样芽孢杆菌、枯草芽孢杆菌、产气荚膜杆菌、大肠杆菌、肺炎克雷伯氏菌、草分枝杆菌、绿脓杆菌、金黃葡萄球菌、奥拉尼堡沙门氏菌、宋内志贺氏菌和化脓性链球菌。

本试剂盒的工作原理在于：SYTO 9和PI的光谱特征以及穿透健康细菌细胞的能力不同。单独使用时，SYTO 9能对群体内的所有细菌进行标记—具有完整膜和受损膜的细菌；相反，PI只能渗透进入受损的膜，PI的插入会引起SYTO 9染色荧光的降低，当体系内加入两种染料时。因此，通过适量比例的SYTO 9和PI的混合染色，具有完整膜结构的细菌呈绿色荧光，而具受损膜结构的细菌呈红色荧光。两者染料的最大激发和发射波长分别是480/500nm（SYTO 9）和490/635nm（PI）。背景基本无荧光。本试剂盒兼容于荧光显微镜，荧光光度计、荧光酶标仪、流式细胞仪或其它荧光检测仪器。

试剂盒组分

保存与运输方法

保存：-20℃避光保存，有效期一年。

运输：冰袋运输。

注意事项

1）    由于试剂盒内SYTO 9和PI的组分量少，室温回温充分融化后，务必低速离心沉至管底后再开盖。

2）    第一次使用可将SYTO 9和PI根据单次用量分装保存，密封后置于≤-20℃避光保存。

3）    组分C（Mounting oil）用于将细菌固定在膜上，25℃的折射率是1.517 ± 0.003。不要用作浸油（Immersion oil）。

4）    SYTO 9和PI结合核酸，PI是潜在的诱变剂，目前没有数据阐明SYTO 9的诱变性或毒性，两种试剂使用都需做恰当防护。DMSO能促进有机分子进入组织。强烈建议处理DMSO储存液时戴双层手套。对于核酸染料，含此类染料的试剂经活性炭吸附后再进行废液处理。活性炭之后经焚烧来破坏染料。

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

以下步骤仅用作示例以指导科研人员开展自身细菌样本的染色。

一、培养条件和细菌悬液的制备

【注意】：用本试剂盒进行细菌染色，务必要小心去除培养基残留，因为，核酸和其它培养基成分可能以不可预料的方式与SYTO 9和PI结合，导致染色结果发生不可接受的变动。简单的一次清洗步骤通常足以去除培养基内含的培养基成分干扰物残留。不建议使用磷酸盐清洗缓冲液，因此可能降低染色效率。

1.1 用营养肉汤培养大肠杆菌或金黄se葡萄球菌（30ml）使其生长至对数生长后期。

1.2 于10000×g离心10-15min，浓缩25ml细菌培养物。

1.3 吸走上清液，用2ml 0.85% NaCl或适当缓冲液来重悬沉淀。

1.4 取1ml重悬菌液分别加入含20ml 0.85% NaCl或适当缓冲液的30-40ml离心管（用作活细菌），或含20ml 70%异丙醇的30-40ml离心管（用作杀死细菌）。

1.5 两管样品于室温孵育1h，每隔15min颠倒混匀一次。

1.6 两管样品于10000×g离心10-15min。

1.7 用20ml 0.85% NaCl或适当缓冲液重悬沉淀，并且按照步骤1.6再离心一次。

1.8 分别用10ml 0.85% NaCl或适当缓冲液重悬两管样品。

1.9 分别取3ml菌液测定670nm的光密度（OD670），用玻璃或丙烯酸酯比色皿（1cm路径）。

1.10 对于大肠杆菌或金黄se葡萄球菌的建议染色浓度，根据你的仪器类型（荧光显微镜、荧光光度经、荧光酶标仪）或流式细胞仪来参考相应部分的染色条件。

二、染色条件的优化

试剂盒内的两种染料都经过平衡优化，按照1：1的比例进行混合用于绝大多数的样本都能得到良好的区分活/死细菌。偶然情况下，两种染料的混合比例需根据实际需求进行优化调整。比如：在待检样本中，绿色荧光太突出，建议要么降低SYTO 9浓度，要么提高PI浓度。

为了全面优化染色条件，建议测试梯度浓度的SYTO 9，每一种浓度与梯度浓度的PI进行组合染色。建议按照1ml细菌悬液加入3µl不同混合比率的染料预混液。

三、荧光显微镜操作步骤

活菌和死菌的荧光可能用标准的荧光素长通滤片设置来同时观察。替代方案的话，活菌（绿色荧光）和死菌（红色荧光）可分别用荧光素和Texas Red带通滤光片设置。用于本试剂盒检测的建议荧光显微镜滤片设置见表1。

表1适用于本试剂盒检测用的常见滤光片特征

3.1 在微量离心管内组合等量的组分A（SYTO 9）和组分B（PI），混匀。

3.2 每1ml细菌悬液内加入3μl染料预混液。按照建议的稀释倍数，最终得到的染色工作液内含0.3% DMSO。更高浓度的DMSO可能对染色产生副效果。

3.3 混匀后室温避光孵育15min。

3.4 吸5μl染色的细菌悬液到载玻片上，并盖上18mm方形盖玻片。

3.5 根据表1选择荧光显微镜上合适的滤片来观察。

四、荧光光度计操作步骤

4.1 调整大肠杆菌悬液（活和杀死）使其密度为1×108个细菌/ml（~0.03 OD670）或金黄se葡萄球菌悬液（活和杀死）使其密度为1×107个细菌/ml（~0.15 OD670）。用于荧光光度计检测，金黄se葡萄球菌悬液的浓度通常比大肠杆菌少10倍。

4.2 参考表2在1cm 玻璃、丙烯酸酯或石英荧光比色皿混匀五种不同比例的细菌悬液。每个样本的总体积为3ml。

表2.荧光光度计法检测活/死细菌所需不同比例活细菌和死细菌悬液的加量体积

4.3 在微量离心管内分别加30μl组分A（SYTO 9）和30μl组分B（PI），混匀。

4.4 每组不同比例的细菌悬液内加入9μl染料预混液（5个样本×9μl =45μl总量），用枪上下吹打数次使其混匀。

4.5 室温避光孵育15min。

4.6荧光测定和数据分析

①用荧光光度计测定每组细菌悬液（Fcell）的荧光发射光谱（激发：470nm，发射：490-700nm）；

②分别测定发射光谱在510-540nm（em1，绿色）和620-650nm（em2，红色）的累积荧光，并计算累积荧光比值：RatioG/R=Fcell,em1/Fcell,em2

③以大肠杆菌悬液内活细胞的占比为横坐标，以累积绿色荧光与红色荧光比（RatioG/R）为纵坐标，制图。

五、荧光酶标仪操作步骤

针对细菌悬液，用荧光酶标仪的测定条件与荧光光度计的基本类似。如同荧光光度计的检测步骤，染料浓度相同于荧光显微镜的建议浓度，绿/红荧光比与活细菌相对数量呈正比。

5.1 调整大肠杆菌悬液（活和杀死）使其密度为2×108个细菌/ml（~0.06 OD670）或金黄se葡萄球菌悬液（活和杀死）使其密度为2×107个细菌/ml（~0.3 OD670）。用于荧光酶标仪检测，金黄se葡萄球菌悬液的浓度通常比大肠杆菌少10倍。

5.2 参考表3在16×125mm高硼硅玻璃培养管内混匀五种不同比例的细菌悬液（大肠杆菌或金黄se葡萄球菌）。每个样本的总体积为2ml。

表3.荧光酶标仪法检测活/死细菌所需不同比例活细菌和死细菌悬液的加量体积

5.3 在微量离心管内分别加6μl组分A（SYTO 9）和6μl组分B（PI），混匀。

5.4 通过将所有的12μl上述预混液加入2ml无菌的dH2O，混匀后制备2×染色混合液。

5.5 吸100μl细菌悬液混合物到平底96孔板的各孔内，建议每个制备物做三个平行。96孔板的边缘孔通常空置以避免假读数。

5.6 更换新的枪头，每孔加入100μl 2×染色混合液，上下吹打使充分混匀。

5.7 室温避光孵育15min。

5.8荧光测定和数据分析

①以~485nm为激发波长，~530nm为发射波长（emission 1，绿色）来测定每孔荧光；

②以~485nm为激发波长，~630nm为发射波长（emission 2，红色）来测定每孔荧光；

③通过测定两种发射波长下的荧光强光，并计算荧光比值：RatioG/R=Fcell,em1/Fcell,em2

④以大肠杆菌悬液内活细胞的占比为横坐标，以RatioG/R为纵坐标，制图。

引用文献：

[1] Wang, H.; Li, Y.; Li, Z.; Ma, R.; Bai, X.; Zhan, X.; Luo, K.; Su, R.; Li, X.; Xia, X.; Shi, C. Inhibition of Cronobacter sakazakii by Litsea cubeba Essential Oil and the Antibacterial Mechanism. Foods 2022, 11, 3900. -------doi.org/10.3390/foods11233900

Cronobacter sakazakii ATCC 29004 suspensions (~10⁸ CFU/mL) were treated with LC-EO (0, 1/4, 1/2, and 1MIC, respectively) at 37 °C for 30 min. After treatment, these samples were centrifuged (10,000× g, 2 min, 4 °C), and the cell pellets were resuspended in 2 mL of 0.85% (w/v) NaCl solution. A live/dead bacterial double stain kit (Shanghai Maokang Biotechnology Co., Ltd., Shanghai, China) was used, and, in accordance with the manufacturer’s instructions, 1.5 μL of fluorescent dyes SYTO 9 and 1.5 μL of propidium iodide (PI) were prepared and mixed. Then, 3 µL of the mixed reagent was added to a 1 mL aliquot of each cell suspension, and cultured in the dark for 10 min at room temperature. The samples were observed via confocal laser scanning microscopy (CLSM; A1, Nikon, Tokyo, Japan) under 300× magnification.

Figure 3. Integrity of the cell membrane of C. sakazakii treated with LC-EO observed by CLSM: (A) 0 (Control); (B) 1/4MIC; (C) 1/2MIC; (D)MIC.

[2]  Liu J, Zhu W, Qin N, Ren X, Xia X. Propionate and Butyrate Inhibit Biofilm Formation of Salmonella Typhimurium Grown in Laboratory Media and Food Models. Foods. 2022 Nov 3;11(21):3493. doi: 10.3390/foods11213493. PMID: 36360105; PMCID: PMC9654251.

S. Typhimurium SL1344 was grown on the glass slides at 37 °C for 24 h in the presence of 1 mg/mL of propionate or butyrate. The untreated S. Typhimurium SL1344 was used as a control. After incubation for 24 h, planktonic bacteria were removed, and biofilm cells were washed twice with 0.01 M PBS. The formed biofilm was stained using SYTO 9/PI live/dead bacterial double stain kit (Maokang, Shanghai, China). After incubation for 25 min, the unbound colorant was rinsed with 0.01 M PBS twice. The images were visualized by fluorescence microscope (Nikon, Tokyo, Japan) at ×10 magnification. Image J calculated the fluorescence intensities of live and dead cells. The results were represented as % of dead cells.

[3] Zhang L, Yang N, Jin Y, Xu X. Putative inactivation mechanism and germicidal efficacy of induced electric field against Staphylococcus aureus. Food Microbiol. 2023 May;111:104208. doi: 10.1016/j.fm.2022.104208. Epub 2022 Dec 13. PMID: 36681392.

SYTO 9/PI Live/Dead Bacterial Double Stain Kit (MX4234-40T; Maokang Biotechnology Co., Ltd, Shanghai, China) was used to assess the cell membrane integrity of S. aureus.

[4] Ziyue Wang et al. Natural biomass-derived carbon dots as potent antimicrobial agents against multidrug-resistant bacteria and their biofilms.  Sustainable Materials and Technologies Volume 36, July 2023, e00584

SYTO 9/PI Live/Dead Bacterial Double Stain Kit purchased from Maokang Biotechnology Co., (Shanghai, China).

[5] Guo L, Ding J, Zhou W. Converting bacteria into autologous tumor vaccine via surface biomineralization of calcium carbonate for enhanced immunotherapy. Acta Pharm Sin B. 2023 Dec;13(12):5074-5090. doi: 10.1016/j.apsb.2023.08.028. Epub 2023 Sep 1. PMID: 38045045; PMCID: PMC10692385.

The calcium ionophore A23187 (MZ2153) and SYTO 9/PI Live/Dead Bacterial Double Stain Kit (MX4234) were purchased from Maokang Biotechnology Co., Ltd. (Shanghai, China).

[6] Chen P, Liu Y, Li C, Hua S, Sun C, Huang L. Antibacterial mechanism of vanillin against Escherichia coli O157: H7. Heliyon. 2023 Aug 19;9(9):e19280. doi: 10.1016/j.heliyon.2023.e19280. PMID: 37662745; PMCID: PMC10474422.

The SYTO 9/PI Live/Dead Bacteria Dual Staining Kit (Shanghai Maokang Biotechnology Co., Ltd., China) was used to detect E. coli O157:H7 necrosis.

[7] Pius Bassey A, Pei Liu P, Chen J, Kabir Bako H, Frimpong Boateng E, Isaiah Ibeogu H, Ye K, Li C, Zhou G. Antibacterial efficacy of phenyllactic acid against Pseudomonas lundensis and Brochothrix thermosphacta and its synergistic application on modified atmosphere/air-packaged fresh pork loins. Food Chem. 2024 Jan 1;430:137002. doi: 10.1016/j.foodchem.2023.137002. Epub 2023 Jul 26. PMID: 37524609.

The viability of the bacterial cells was assessed by Syto 9/PI Live/ Dead Bacterial Double Stain Kit (Maokang Biotechnology Co., Ltd., Shanghai, China) according….

[8] Zhao Y, Chen G, Yushanjiang S, Zhao M, Yang H, Lu R, Qu R, Dai Y, Yang L. In vitro and in vivo study of antibacterial and anti-encrustation coating on ureteric stents. BJU Int. 2024 Mar 8. doi: 10.1111/bju.16326. Epub ahead of print. PMID: 38459675.

A SYTO 9/propidium iodide (PI) Live/Dead Bacterial Double Stain Kit was purchased from MaoKang Biotechnology Corporation, Shanghai, China.

[9] Liu T, Liu W, Zeng L, Wen Z, Xiong Z, Liao Z, Hu Y. Biofunctionalization of 3D Printed Porous Tantalum Using a Vancomycin-Carboxymethyl Chitosan Composite Coating to Improve Osteogenesis and Antibiofilm Properties. ACS Appl Mater Interfaces. 2022 Sep 21;14(37):41764-41778. doi: 10.1021/acsami.2c11715. Epub 2022 Sep 10. PMID: 36087275.

[10] Liufang Zhou et al. Effect of poly(styrene sulfonate) treatment on the structural evolution and sonodynamic performance of PCN-224 nanoparticles. Colloids and Surfaces A: Physicochemical and Engineering Aspects. Volume 688, 5 May 2024, 133603

SYTO 9/PI live /dead bacterial double stain kit was purchased from Shanghai Maokang Biotechnology Co., LTD.

[11] Qin X, Wu Y, Zhao Y, Qin S, Ji Q, Jia J, Huo M, Zhao X, Ma Q, Wang X, Chen X, Zhang H, Zhang M, Yang L, Li W, Tang J. Revealing active constituents within traditional Chinese Medicine used for treating bacterial pneumonia, with emphasis on the mechanism of baicalein against multi-drug resistant Klebsiella pneumoniae. J Ethnopharmacol. 2024 Mar 1;321:117488. doi: 10.1016/j.jep.2023.117488. Epub 2023 Nov 25. PMID: 38008277.

The suspension was discarded and stained according to the instructions of SYTO 9/PI Live/Dead Bacterial Double Stain Kit (Shanghai Maokang Biotechnology Co., Ltd. Lot: 2002X230160).

[12] Liang F, Liu X, Yu X, Liu L, He H, Huang C, Hu J, Wang Z, Zhou Y, Zhai Y. Enhancing bioavailable carbon sources and minimizing ammonia emissions in distillery sludge and distiller's grains waste co-composting through deep eutectic solvent addition. Bioresour Technol. 2024 Apr;397:130491. doi: 10.1016/j.biortech.2024.130491. Epub 2024 Feb 24. PMID: 38408502.

The live/dead bacterial ratio evaluation was conducted using the SYTO 9/PI dual-staining bacterial viability kit (MX4234-40T; Maokang Biotechnology Co

[13] Zhang Y, Zheng M, Wang Z, Liu Z, Chen S, Li X, Shi Y, Hu H. Discovery of novel antibacterial agent for the infected wound treatment: all-hydrocarbon stapling optimization of LL-37. Theranostics. 2024 Jan 20;14(3):1181-1194. doi: 10.7150/thno.87916. PMID: 38323312; PMCID: PMC10845205.

According to the instructions from manufacturer, membrane permeability was measured utilizing the SYTO 9/PI Live/Dead Bacterial Double Staining Kit (Maokang, Shanghai, China).

[14] Kaixin Yi et al. Semi-interpenetrating network hydrogels-based microcapsule for quorum quenching bacteria biocontainment to enhance biofouling control in membrane bioreactor. Chemical Engineering Journal. Volume 486, 15 April 2024, 150103

And biofilm on the membrane surface was stained with MKBio SYTO 9/PI live/dead bacteria double stain kit (Maokang Biotechnology, Shanghai) for 15 min in the dark

[15] Xie LY, Xu YB, Ding XQ, Liang S, Li DL, Fu AK, Zhan XA. Itaconic acid and dimethyl itaconate exert antibacterial activity in carbon-enriched environments through the TCA cycle. Biomed Pharmacother. 2023 Nov;167:115487. doi: 10.1016/j.biopha.2023.115487. Epub 2023 Sep 15. PMID: 37713987.

The live or dead status of the bacteria was observed using a LIVE/DEAD viability kit containing green-fluorescent Syto 9 and red-fluorescent PI (Maokangbio, Shanghai, China).

[16] Liu L, Li S, Yang K, Chen Z, Li Q, Zheng L, Wu Z, Zhang X, Su L, Wu Y, Song J. Drug-Free Antimicrobial Nanomotor for Precise Treatment of Multidrug-Resistant Bacterial Infections. Nano Lett. 2023 May 10;23(9):3929-3938. doi: 10.1021/acs.nanolett.3c00632. Epub 2023 Apr 27. PMID: 37129144.

MKBio SYTO 9/PI Live/Dead Bacterial Double Stain Kit was purchased from Shanghai Maokang Biotechnology Co., LTD.

Yufang Tan et al. Degradable Microneedle Patch Loaded with Doxycycline Hydrochloride and Vascular Endothelial Growth Factors for Promoting Diabetic Wound Healing.

— —Written/Edited by V. Shallan【版权归MKBio懋康所有】

上海懋康生物科技有限公司是一家涉足于生命科学和生物技术领域研究的试剂、仪器和实验室消耗品与实验服务工作，主要从事细胞生物学、植物学、分子生物学、免疫学、生物化学、蛋白组学。生物制药与诊断试剂研发生产等领域。 本公司秉承“以人为本，以诚为信、合同守信”的经营理念。坚持"品质保障"的原则为广大客户提供优质产品。

活细菌/死细菌双染试剂盒荧光探针

活细菌/死细菌双染试剂盒荧光探针