SYTO 9/PI Live/Dead Bacterial Double Stain Kit

活細菌/死細菌雙染試劑盒

産品信息

試劑盒規格說明（以MX4234-40T為(wèi)例，按照(zhào)建議的試劑稀釋倍數和單次測試體積來計算(suàn)）：

◇ 熒光(guāng)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次獨立測試。

◇ 熒光(guāng)酶标儀檢測，2ml無菌水(shuǐ)加入12μl染料混合液（SYTO 6.0μl +PI 6.0μl），制備成2×工(gōng)作液。按照(zhào)100μl染料混合液加入100μl菌液的比例使用，可做200次獨立測試。

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

産品描述

活細菌/死細菌雙染試劑盒（SYTO 9/PI Live/Dead Bacterial Double Stain Kit）是一(yī)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(hóng)色熒光(guāng)核酸染料來進行細菌活力的檢測，适用于大量的細菌種屬，包括蠟樣芽孢杆菌、枯草(cǎo)芽孢杆菌、産氣莢膜杆菌、大腸杆菌、肺炎克雷伯氏菌、草(cǎo)分枝杆菌、綠膿杆菌、金黃葡萄球菌、奧拉尼堡沙門(mén)氏菌、宋内志(zhì)賀氏菌和化膿性鏈球菌。

本試劑盒的工(gōng)作原理在于：SYTO 9和PI的光(guāng)譜特征以及穿透健康細菌細胞的能(néng)力不同。單獨使用時，SYTO 9能(néng)對群體内的所有細菌進行标記—具有完整膜和受損膜的細菌；相(xiàng)反，PI隻能(néng)滲透進入受損的膜，PI的插入會(huì)引起SYTO 9染色熒光(guāng)的降低(dī)，當體系内加入兩種染料時。因此，通(tōng)過适量比例的SYTO 9和PI的混合染色，具有完整膜結構的細菌呈綠色熒光(guāng)，而具受損膜結構的細菌呈紅(hóng)色熒光(guāng)。兩者染料的最大激發和發射波長(cháng)分别是480/500nm（SYTO 9）和490/635nm（PI）。背景基本無熒光(guāng)。本試劑盒兼容于熒光(guāng)顯微鏡，熒光(guāng)光(guāng)度計、熒光(guāng)酶标儀、流式細胞儀或其它熒光(guāng)檢測儀器(qì)。

試劑盒組分

保存與運輸方法

保存：-20℃避光(guāng)保存，有效期一(yī)年(nián)。

運輸：冰袋運輸。

注意事(shì)項

1）    由于試劑盒内SYTO 9和PI的組分量少，室溫回溫充分融化後，務必低(dī)速離心沉至管底後再開(kāi)蓋。

2）    第一(yī)次使用可将SYTO 9和PI根據單次用量分裝保存，密封後置于≤-20℃避光(guāng)保存。

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

4）    SYTO 9和PI結合核酸，PI是潛在的誘變劑，目前沒有數據闡明SYTO 9的誘變性或毒性，兩種試劑使用都需做恰當防護。DMSO能(néng)促進有機(jī)分子進入組織。強烈建議處理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活性炭吸附後再進行廢液處理。活性炭之後經焚燒來破壞染料。

5）    為(wèi)了您的安全和健康，請穿實驗服并戴一(yī)次性手套操作。

使用方法

以下(xià)步驟僅用作示例以指導科研人員(yuán)開(kāi)展自(zì)身細菌樣本的染色。

一(yī)、培養條件(jiàn)和細菌懸液的制備

【注意】：用本試劑盒進行細菌染色，務必要小(xiǎo)心去除培養基殘留，因為(wèi)，核酸和其它培養基成分可能(néng)以不可預料的方式與SYTO 9和PI結合，導緻染色結果發生(shēng)不可接受的變動。簡單的一(yī)次清洗步驟通(tōng)常足以去除培養基内含的培養基成分幹擾物(wù)殘留。不建議使用磷酸鹽清洗緩沖液，因此可能(néng)降低(dī)染色效率。

1.1 用營養肉湯培養大腸杆菌或金黃色葡萄球菌（30ml）使其生(shēng)長(cháng)至對數生(shēng)長(cháng)後期。

1.2 于10000×g離心10-15min，濃縮25ml細菌培養物(wù)。

1.3 吸走上(shàng)清液，用2ml 0.85% NaCl或适當緩沖液來重懸沉澱。

1.4 取1ml重懸菌液分别加入含20ml 0.85% NaCl或适當緩沖液的30-40ml離心管（用作活細菌），或含20ml 70%異丙醇的30-40ml離心管（用作殺死細菌）。

1.5 兩管樣品于室溫孵育1h，每隔15min颠倒混勻一(yī)次。

1.6 兩管樣品于10000×g離心10-15min。

1.7 用20ml 0.85% NaCl或适當緩沖液重懸沉澱，并且按照(zhào)步驟1.6再離心一(yī)次。

1.8 分别用10ml 0.85% NaCl或适當緩沖液重懸兩管樣品。

1.9 分别取3ml菌液測定670nm的光(guāng)密度（OD670），用玻璃或丙烯酸酯比色皿（1cm路(lù)徑）。

1.10 對于大腸杆菌或金黃色葡萄球菌的建議染色濃度，根據你的儀器(qì)類型（熒光(guāng)顯微鏡、熒光(guāng)光(guāng)度經、熒光(guāng)酶标儀）或流式細胞儀來參考相(xiàng)應部分的染色條件(jiàn)。

二、染色條件(jiàn)的優化

試劑盒内的兩種染料都經過平衡優化，按照(zhào)1：1的比例進行混合用于絕大多(duō)數的樣本都能(néng)得到(dào)良好的區分活/死細菌。偶然情況下(xià)，兩種染料的混合比例需根據實際需求進行優化調整。比如：在待檢樣本中，綠色熒光(guāng)太突出，建議要麽降低(dī)SYTO 9濃度，要麽提高(gāo)PI濃度。

為(wèi)了全面優化染色條件(jiàn)，建議測試梯度濃度的SYTO 9，每一(yī)種濃度與梯度濃度的PI進行組合染色。建議按照(zhào)1ml細菌懸液加入3µl不同混合比率的染料預混液。

三、熒光(guāng)顯微鏡操作步驟

活菌和死菌的熒光(guāng)可能(néng)用标準的熒光(guāng)素長(cháng)通(tōng)濾片設置來同時觀察。替代方案的話，活菌（綠色熒光(guāng)）和死菌（紅(hóng)色熒光(guāng)）可分别用熒光(guāng)素和Texas Red帶通(tōng)濾光(guāng)片設置。用于本試劑盒檢測的建議熒光(guāng)顯微鏡濾片設置見(jiàn)表1。

表1适用于本試劑盒檢測用的常見(jiàn)濾光(guāng)片特征

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

3.2 每1ml細菌懸液内加入3μl染料預混液。按照(zhào)建議的稀釋倍數，最終得到(dào)的染色工(gōng)作液内含0.3% DMSO。更高(gāo)濃度的DMSO可能(néng)對染色産生(shēng)副效果。

3.3 混勻後室溫避光(guāng)孵育15min。

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

3.5 根據表1選擇熒光(guāng)顯微鏡上(shàng)合适的濾片來觀察。

四、熒光(guāng)光(guāng)度計操作步驟

4.1 調整大腸杆菌懸液（活和殺死）使其密度為(wèi)1×108個(gè)細菌/ml（~0.03 OD670）或金黃色葡萄球菌懸液（活和殺死）使其密度為(wèi)1×107個(gè)細菌/ml（~0.15 OD670）。用于熒光(guāng)光(guāng)度計檢測，金黃色葡萄球菌懸液的濃度通(tōng)常比大腸杆菌少10倍。

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

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

4.3 在微量離心管内分别加30μl組分A（SYTO 9）和30μl組分B（PI），混勻。

4.4 每組不同比例的細菌懸液内加入9μl染料預混液（5個(gè)樣本×9μl =45μl總量），用槍上(shàng)下(xià)吹打數次使其混勻。

4.5 室溫避光(guāng)孵育15min。

4.6熒光(guāng)測定和數據分析

①用熒光(guāng)光(guāng)度計測定每組細菌懸液（Fcell）的熒光(guāng)發射光(guāng)譜（激發：470nm，發射：490-700nm）；

②分别測定發射光(guāng)譜在510-540nm（em1，綠色）和620-650nm（em2，紅(hóng)色）的累積熒光(guāng)，并計算(suàn)累積熒光(guāng)比值：RatioG/R=Fcell,em1/Fcell,em2

③以大腸杆菌懸液内活細胞的占比為(wèi)橫坐标，以累積綠色熒光(guāng)與紅(hóng)色熒光(guāng)比（RatioG/R）為(wèi)縱坐标，制圖。

五、熒光(guāng)酶标儀操作步驟

針對細菌懸液，用熒光(guāng)酶标儀的測定條件(jiàn)與熒光(guāng)光(guāng)度計的基本類似。如同熒光(guāng)光(guāng)度計的檢測步驟，染料濃度相(xiàng)同于熒光(guāng)顯微鏡的建議濃度，綠/紅(hóng)熒光(guāng)比與活細菌相(xiàng)對數量呈正比。

5.1 調整大腸杆菌懸液（活和殺死）使其密度為(wèi)2×108個(gè)細菌/ml（~0.06 OD670）或金黃色葡萄球菌懸液（活和殺死）使其密度為(wèi)2×107個(gè)細菌/ml（~0.3 OD670）。用于熒光(guāng)酶标儀檢測，金黃色葡萄球菌懸液的濃度通(tōng)常比大腸杆菌少10倍。

5.2 參考表3在16×125mm高(gāo)硼矽玻璃培養管内混勻五種不同比例的細菌懸液（大腸杆菌或金黃色葡萄球菌）。每個(gè)樣本的總體積為(wèi)2ml。

表3.熒光(guāng)酶标儀法檢測活/死細菌所需不同比例活細菌和死細菌懸液的加量體積

5.3 在微量離心管内分别加6μl組分A（SYTO 9）和6μl組分B（PI），混勻。

5.4 通(tōng)過将所有的12μl上(shàng)述預混液加入2ml無菌的dH2O，混勻後制備2×染色混合液。

5.5 吸100μl細菌懸液混合物(wù)到(dào)平底96孔闆的各孔内，建議每個(gè)制備物(wù)做三個(gè)平行。96孔闆的邊緣孔通(tōng)常空置以避免假讀(dú)數。

5.6 更換新的槍頭，每孔加入100μl 2×染色混合液，上(shàng)下(xià)吹打使充分混勻。

5.7 室溫避光(guāng)孵育15min。

5.8熒光(guāng)測定和數據分析

①以~485nm為(wèi)激發波長(cháng)，~530nm為(wèi)發射波長(cháng)（emission 1，綠色）來測定每孔熒光(guāng)；

②以~485nm為(wèi)激發波長(cháng)，~630nm為(wèi)發射波長(cháng)（emission 2，紅(hóng)色）來測定每孔熒光(guāng)；

③通(tōng)過測定兩種發射波長(cháng)下(xià)的熒光(guāng)強光(guāng)，并計算(suàn)熒光(guāng)比值：RatioG/R=Fcell,em1/Fcell,em2

④以大腸杆菌懸液内活細胞的占比為(wèi)橫坐标，以RatioG/R為(wèi)縱坐标，制圖。

引用文獻：

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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【版權歸MKBio懋康所有】

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