acinetobacter biofilm quantification using crystal violet

アシネトバクターのバイオフィルム形成の評価とバイオフィルムの特性評価

Acinetobacter is known to cause nosocomial infections, and its human infection, especially in healthcare facilities, is increasingly reported1. It is widespread in hospitals, healthcare facilities, and food-associated environments2,3,4. It can survive for a long period in environments including hospital surfaces such as bed rails, bedside tables, the surface of ventilators, and sinks4. Such persistence on environmental surfaces may be one of the significant factors contributing to the nosocomial infections of Acinetobacter4.
Biofilm is a form of microbial life and is a microbial matrix composed of live microbial cells and extracellular polymeric substances (EPS) from the cells5. The microbial cells are embedded in the matrix and are often highly resistant to environmental stresses such as heat, salts, dryness, antibiotics, disinfectants, and shear forces6,7.
Acinetobacter can form biofilms on surfaces, suggesting that it may contribute to extended survival on environmental surfaces, including hospital surfaces, and enhanced resistance to antibiotic treatment8,9. In addition, the biofilm formation of Acinetobacter could be highly associated with human clinical outcomes8. Therefore, the biofilm formability of Acinetobacter strains could be one of the indicators in predicting environmental survival and human infections8,10.
The surface-attached biofilms can be quantified and visualized to assess biofilm formability. To quantify surface-attached biofilms, the biofilms are normally stained by biofilm-staining dyes such as crystal violet, and the dyes are eluted in solution and measured for optical density11. Visualization of biofilms is another good approach to assess biofilm formability11. Confocal Laser Scanning Microscopy (CLSM) visualization method employing specificity using fluorescent dyes could be more useful to characterize biofilm morphology compared to other techniques such as SEM12,13.
The viable cells in biofilms can be counted to estimate the number of viable cells in biofilms11. The viable cells embedded in biofilms are detached, diluted, spread on agar plates, incubated, and enumerated. Because the higher number of cells is likely to meet the infectious dose, it can provide more detailed information on biofilms, such as infection potentials associated with the number of cells13,14.
This article presents step-by-step protocols to (1) quantify surface-attached biofilms, (2) count viable cells in the biofilms, and (3) visualize the biofilms using CLSM of Acinetobacter. The presented protocols describe the methods to assess the biofilm formability of Acinetobacter isolates and characterize their biofilms.

著者スポットライト:アシネトバクターバイオフィルムの定量化、評価、および視覚化のための包括的なプロトコル 

アシネトバクターバイオフィルムの定量化、生存率評価、可視化戦略

Watch this Scientific Journal Video about Acinetobacter Biofilm Quantification Using Crystal Violet at JoVE.com

Acinetobacter Biofilm Quantification Using Crystal Violet  

アシネトバクター Crystal Violetを用いたバイオフィルム定量

まず、滅菌ポリスチレン96ウェルプレートの各ウェルに200マイクロリットルの細菌接種物を追加します。次に、200マイクロリットルの脱イオン水を最も外側の井戸に追加して、内側の井戸が乾燥しないようにします。96ウェルプレートを摂氏25度で24時間インキュベートします。インキュベーション後、ピペットを使用して各ウェルで上清を廃棄します。各ウェルに300マイクロリットルのPBSを慎重に加え、ピペットを使用してPBSを再度除去します。200マイクロリットルの1%クリスタルバイオレット溶液を各ウェルに加え、プレートを室温で30分間インキュベートします。次に、各ウェルに200マイクロリットルの無水エタノールを分注した後、プレートを室温で15分間放置します。各ウェルの内容物をピペッティングで繰り返し上下に混合し、完全に混合します。マイクロプレートリーダーを使用して、595ナノメートルでウェルの吸光度を測定し、バイオフィルムの質量を決定します。バイオフィルムの数は、0.04から1.69の光学濃度またはOD値の範囲で、株によって大きく異なり、A.bouvetiiを除くすべてのアセトバクター分離株はバイオフィルムを形成し、A.radioresistensは弱いバイオフィルムを形成しました。A.junii と A.baumannii は中程度のバイオフィルムを形成し、A.pittii と A.ursingii は強いバイオフィルムを形成した。

acinetobacter-biofilm-quantification-using-crystal-violet

Research

JoVE Journal

Biology

Acinetobacter Biofilm Quantification Using Crystal Violet

554 ビュー. まず、滅菌ポリスチレン96ウェルプレートの各ウェルに200マイクロリットルの細菌接種物を追加します。次に、200マイクロリットルの脱イオン水を最も外側の井戸に追加して、内側の井戸が乾燥しないようにします。96ウェルプレートを摂氏25度で24時間インキュベートします。インキュベーション後、ピペットを使用して各ウェルで上清を廃棄します。各ウェルに30...

ビデオ: Acinetobacter Biofilm Quantification Using Crystal Violet  

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, biofilm quantification and visualization are important methods to assess the potential of Acinetobacter strains to cause nosocomial infections. The biofilms forming on the surface of the microplate can be quantified in terms of volume and cell numbers. Biofilm volumes can be quantified by staining using crystal violet, washing, destaining using ethanol, then measuring the solubilized dye using a microplate reader. To quantify the number of cells embedded in the biofilms, the biofilms are scrapped off using cell scrapers, harvested in the saline, vigorously agitated in the presence of glass beads, and spread on Acinetobacter agar. Then, the plates are incubated at 30 &#176;C for 24-42 h. After incubation, the red colonies are enumerated to estimate the number of cells in biofilms. This viable count method can also be useful for counting Acinetobacter cells in mixed-species biofilms. Acinetobacter biofilms can be visualized using fluorescent dyes. A commercially available microplate designed for microscopic analysis is employed to form biofilms. Then, the bottom-surface attached biofilms are stained with SYTO9 and propidium iodide dyes, washed, then visualized with confocal laser scanning microscopy.

This protocol describes the preparation of the inoculum, the biofilm quantification on microtiter plates using crystal violet dye, the viable count in biofilms, and the visualization of biofilms of Acinetobacter.

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...