SYTO 9/PI Live/Dead Bacterial Double Stain Kit

活細(xì)菌/死細(xì)菌雙染試劑盒

產(chǎn)品信息

試劑盒規(guī)格說明（以MX4234-40T為例，按照建議的試劑稀釋倍數(shù)和單次測試體積來計算）：

◇ 熒光顯微鏡檢測，1ml菌液加入3μl染料混合液（SYTO 1.5μl +PI 1.5μl），可做40次獨(dú)立測試。

◇ 熒光酶標(biāo)儀檢測，2ml無菌水加入12μl染料混合液（SYTO 6.0μl +PI 6.0μl），制備成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次獨(dú)立測試。

◇ 流式細(xì)胞儀檢測，2ml菌液加入6μl染料混合液（SYTO 3.0μl +PI 3.0μl），可做20次獨(dú)立測試。

產(chǎn)品描述

活細(xì)菌/死細(xì)菌雙染試劑盒（SYTO 9/PI Live/Dead Bacterial Double Stain Kit）是一款方便且操作簡單的試劑盒，利用SYTO 9綠色核酸染料和碘化丙啶（PI）紅色熒光核酸染料來進(jìn)行細(xì)菌活力的檢測，適用于大量的細(xì)菌種屬，包括蠟樣芽孢桿菌、枯草芽孢桿菌、產(chǎn)氣莢膜桿菌、大腸桿菌、肺炎克雷伯氏菌、草分枝桿菌、綠膿桿菌、金黃葡萄球菌、奧拉尼堡沙門氏菌、宋內(nèi)志賀氏菌和化膿性鏈球菌。

本試劑盒的工作原理在于：SYTO 9和PI的光譜特征以及穿透健康細(xì)菌細(xì)胞的能力不同。單獨(dú)使用時，SYTO 9能對群體內(nèi)的所有細(xì)菌進(jìn)行標(biāo)記—具有完整膜和受損膜的細(xì)菌；相反，PI只能滲透進(jìn)入受損的膜，PI的插入會引起SYTO 9染色熒光的降低，當(dāng)體系內(nèi)加入兩種染料時。因此，通過適量比例的SYTO 9和PI的混合染色，具有完整膜結(jié)構(gòu)的細(xì)菌呈綠色熒光，而具受損膜結(jié)構(gòu)的細(xì)菌呈紅色熒光。兩者染料的激發(fā)和發(fā)射波長分別是480/500nm（SYTO 9）和490/635nm（PI）。背景基本無熒光。本試劑盒兼容于熒光顯微鏡，熒光光度計、熒光酶標(biāo)儀、流式細(xì)胞儀或其它熒光檢測儀器。

試劑盒組分

保存與運(yùn)輸方法

保存：-20℃避光保存，有效期一年。

運(yùn)輸：冰袋運(yùn)輸。

注意事項(xiàng)

1）    由于試劑盒內(nèi)SYTO 9和PI的組分量少，室溫回溫充分融化后，務(wù)必低速離心沉至管底后再開蓋。

2）    次使用可將SYTO 9和PI根據(jù)單次用量分裝保存，密封后置于≤-20℃避光保存。

3）    組分C（Mounting oil）用于將細(xì)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結(jié)合核酸，PI是潛在的誘變劑，目前沒有數(shù)據(jù)闡明SYTO 9的誘變性或毒性，兩種試劑使用都需做恰當(dāng)防護(hù)。DMSO能促進(jìn)有機(jī)分子進(jìn)入組織。強(qiáng)烈建議處理DMSO儲存液時戴雙層手套。對于核酸染料，含此類染料的試劑經(jīng)活性炭吸附后再進(jìn)行廢液處理?；钚蕴恐蠼?jīng)焚燒來破壞染料。

5）    為了您的安全和健康，請穿實(shí)驗(yàn)服并戴一次性手套操作。

使用方法

以下步驟僅用作示例以指導(dǎo)科研人員開展自身細(xì)菌樣本的染色。

一、培養(yǎng)條件和細(xì)菌懸液的制備

【注意】：用本試劑盒進(jìn)行細(xì)菌染色，務(wù)必要小心去除培養(yǎng)基殘留，因?yàn)?，核酸和其它培養(yǎng)基成分可能以不可預(yù)料的方式與SYTO 9和PI結(jié)合，導(dǎo)致染色結(jié)果發(fā)生不可接受的變動。簡單的一次清洗步驟通常足以去除培養(yǎng)基內(nèi)含的培養(yǎng)基成分干擾物殘留。不建議使用磷酸鹽清洗緩沖液，因此可能降低染色效率。

1.1 用營養(yǎng)肉湯培養(yǎng)大腸桿菌或金黃se葡萄球菌（30ml）使其生長至對數(shù)生長后期。

1.2 于10000×g離心10-15min，濃縮25ml細(xì)菌培養(yǎng)物。

1.3 吸走上清液，用2ml 0.85% NaCl或適當(dāng)緩沖液來重懸沉淀。

1.4 取1ml重懸菌液分別加入含20ml 0.85% NaCl或適當(dāng)緩沖液的30-40ml離心管（用作活細(xì)菌），或含20ml 70%異丙醇的30-40ml離心管（用作殺死細(xì)菌）。

1.5 兩管樣品于室溫孵育1h，每隔15min顛倒混勻一次。

1.6 兩管樣品于10000×g離心10-15min。

1.7 用20ml 0.85% NaCl或適當(dāng)緩沖液重懸沉淀，并且按照步驟1.6再離心一次。

1.8 分別用10ml 0.85% NaCl或適當(dāng)緩沖液重懸兩管樣品。

1.9 分別取3ml菌液測定670nm的光密度（OD670），用玻璃或丙烯酸酯比色皿（1cm路徑）。

1.10 對于大腸桿菌或金黃se葡萄球菌的建議染色濃度，根據(jù)你的儀器類型（熒光顯微鏡、熒光光度經(jīng)、熒光酶標(biāo)儀）或流式細(xì)胞儀來參考相應(yīng)部分的染色條件。

二、染色條件的優(yōu)化

試劑盒內(nèi)的兩種染料都經(jīng)過平衡優(yōu)化，按照1：1的比例進(jìn)行混合用于絕大多數(shù)的樣本都能得到良好的區(qū)分活/死細(xì)菌。偶然情況下，兩種染料的混合比例需根據(jù)實(shí)際需求進(jìn)行優(yōu)化調(diào)整。比如：在待檢樣本中，綠色熒光太突出，建議要么降低SYTO 9濃度，要么提高PI濃度。

為了全面優(yōu)化染色條件，建議測試梯度濃度的SYTO 9，每一種濃度與梯度濃度的PI進(jìn)行組合染色。建議按照1ml細(xì)菌懸液加入3µl不同混合比率的染料預(yù)混液。

三、熒光顯微鏡操作步驟

活菌和死菌的熒光可能用標(biāo)準(zhǔn)的熒光素長通濾片設(shè)置來同時觀察。替代方案的話，活菌（綠色熒光）和死菌（紅色熒光）可分別用熒光素和Texas Red帶通濾光片設(shè)置。用于本試劑盒檢測的建議熒光顯微鏡濾片設(shè)置見表1。

表1適用于本試劑盒檢測用的常見濾光片特征

3.1 在微量離心管內(nèi)組合等量的組分A（SYTO 9）和組分B（PI），混勻。

3.2 每1ml細(xì)菌懸液內(nèi)加入3μl染料預(yù)混液。按照建議的稀釋倍數(shù)，最終得到的染色工作液內(nèi)含0.3% DMSO。更高濃度的DMSO可能對染色產(chǎn)生副效果。

3.3 混勻后室溫避光孵育15min。

3.4 吸5μl染色的細(xì)菌懸液到載玻片上，并蓋上18mm方形蓋玻片。

3.5 根據(jù)表1選擇熒光顯微鏡上合適的濾片來觀察。

四、熒光光度計操作步驟

4.1 調(diào)整大腸桿菌懸液（活和殺死）使其密度為1×108個細(xì)菌/ml（~0.03 OD670）或金黃se葡萄球菌懸液（活和殺死）使其密度為1×107個細(xì)菌/ml（~0.15 OD670）。用于熒光光度計檢測，金黃se葡萄球菌懸液的濃度通常比大腸桿菌少10倍。

4.2 參考表2在1cm 玻璃、丙烯酸酯或石英熒光比色皿混勻五種不同比例的細(xì)菌懸液。每個樣本的總體積為3ml。

表2.熒光光度計法檢測活/死細(xì)菌所需不同比例活細(xì)菌和死細(xì)菌懸液的加量體積

4.3 在微量離心管內(nèi)分別加30μl組分A（SYTO 9）和30μl組分B（PI），混勻。

4.4 每組不同比例的細(xì)菌懸液內(nèi)加入9μl染料預(yù)混液（5個樣本×9μl =45μl總量），用槍上下吹打數(shù)次使其混勻。

4.5 室溫避光孵育15min。

4.6熒光測定和數(shù)據(jù)分析

①用熒光光度計測定每組細(xì)菌懸液（Fcell）的熒光發(fā)射光譜（激發(fā)：470nm，發(fā)射：490-700nm）；

②分別測定發(fā)射光譜在510-540nm（em1，綠色）和620-650nm（em2，紅色）的累積熒光，并計算累積熒光比值：RatioG/R=Fcell,em1/Fcell,em2

③以大腸桿菌懸液內(nèi)活細(xì)胞的占比為橫坐標(biāo)，以累積綠色熒光與紅色熒光比（RatioG/R）為縱坐標(biāo)，制圖。

五、熒光酶標(biāo)儀操作步驟

針對細(xì)菌懸液，用熒光酶標(biāo)儀的測定條件與熒光光度計的基本類似。如同熒光光度計的檢測步驟，染料濃度相同于熒光顯微鏡的建議濃度，綠/紅熒光比與活細(xì)菌相對數(shù)量呈正比。

5.1 調(diào)整大腸桿菌懸液（活和殺死）使其密度為2×108個細(xì)菌/ml（~0.06 OD670）或金黃se葡萄球菌懸液（活和殺死）使其密度為2×107個細(xì)菌/ml（~0.3 OD670）。用于熒光酶標(biāo)儀檢測，金黃se葡萄球菌懸液的濃度通常比大腸桿菌少10倍。

5.2 參考表3在16×125mm高硼硅玻璃培養(yǎng)管內(nèi)混勻五種不同比例的細(xì)菌懸液（大腸桿菌或金黃se葡萄球菌）。每個樣本的總體積為2ml。

表3.熒光酶標(biāo)儀法檢測活/死細(xì)菌所需不同比例活細(xì)菌和死細(xì)菌懸液的加量體積

5.3 在微量離心管內(nèi)分別加6μl組分A（SYTO 9）和6μl組分B（PI），混勻。

5.4 通過將所有的12μl上述預(yù)混液加入2ml無菌的dH2O，混勻后制備2×染色混合液。

5.5 吸100μl細(xì)菌懸液混合物到平底96孔板的各孔內(nèi)，建議每個制備物做三個平行。96孔板的邊緣孔通?？罩靡员苊饧僮x數(shù)。

5.6 更換新的槍頭，每孔加入100μl 2×染色混合液，上下吹打使充分混勻。

5.7 室溫避光孵育15min。

5.8熒光測定和數(shù)據(jù)分析

①以~485nm為激發(fā)波長，~530nm為發(fā)射波長（emission 1，綠色）來測定每孔熒光；

②以~485nm為激發(fā)波長，~630nm為發(fā)射波長（emission 2，紅色）來測定每孔熒光；

③通過測定兩種發(fā)射波長下的熒光強(qiáng)光，并計算熒光比值：RatioG/R=Fcell,em1/Fcell,em2

④以大腸桿菌懸液內(nèi)活細(xì)胞的占比為橫坐標(biāo)，以RatioG/R為縱坐標(biāo)，制圖。

引用文獻(xiàn)：

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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.

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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….

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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【版權(quán)歸MKBio懋康所有】

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