Name: Acinetobacter Biofilm Quantification Using Crystal Violet
Uploaded: 2023-08-04T14:10:00
Description: Watch this Scientific Journal Video about Acinetobacter Biofilm Quantification Using Crystal Violet at JoVE.com

acinetobacter biofilm quantification using crystal violet

Bedömning av bildning av acinetobacter biofilm och karakterisering av biofilmer

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.

Författare i fokus: Ett omfattande protokoll för kvantifiering, bedömning och visualisering av biofilm med acinetobacter 

Kvantifiering, viabilitetsbedömning och visualiseringsstrategier för biofilmer av acinetobacter

Biofilms are composed of live microbial cells and are difficult to remove from the surfaces. Acinetobacter strains commonly found in our environment, causing nosocomial infections, are also known to form biofilms on surfaces. So, we aim to develop methods to both quantify and visualize Acinetobacter biofilms.Our protocol provides a simple and easy method to quantify and visualize the biofilms of Acinetobacter. The viable number of cells inside the biofilms is quantified by using the viable count method. Our research findings reveal that the naturally-existing Acinetobacter vector strains have quite varying potentials for biofilm formation.Also, there will exist distinct morphological differences within the various biofilms. This paved the way for research focusing on the causes of such a large variation among Acinetobacter strains.

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

Acinetobacter Biofilm Quantification Using Crystal Violet  

Acinetobacter Kvantifiering av biofilm med hjälp av kristallviolett

Till att börja med, tillsätt 200 mikroliter av bakterieinokulatet till varje brunn på en steril 96-håls polystyrenplatta. Tillsätt sedan 200 mikroliter avjoniserat vatten i de yttersta brunnarna för att förhindra att de inre brunnarna torkar ut. Inkubera plattan med 96 brunnar vid 25 grader Celsius i 24 timmar.Efter inkubationen kasseras supernatanten i varje brunn med hjälp av en pipett. Tillsätt försiktigt 300 mikroliter PBS till varje brunn och ta bort PBS igen med hjälp av pipetten. Tillsätt 200 mikroliter 1% kristallviolett lösning i varje brunn innan du inkuberar plattan i rumstemperatur i 30 minuter.Efter att ha doserat 200 mikroliter absolut etanol i varje brunn, låt plattan stå i 15 minuter i rumstemperatur. Säkerställ noggrann blandning genom att pipettera innehållet i varje brunn upp och ner upprepade gånger. Mät brunnarnas absorbans vid 595 nanometer med hjälp av en mikroplattläsare för att bestämma biofilmmassan.Antalet biofilmer varierade kraftigt beroende på stammarna, från en optisk densitet eller OD-värde på 0,04 till 1,69 Alla Acetobacter-isolat, förutom A. bouvetii, bildade biofilmer, medan A.radioresistens bildade en svag biofilm. A. junii och A. baumannii bildade måttliga biofilmer, och A. pittii och A. ursingii bildade starka biofilmer.

Research

JoVE Journal

Biology

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, ...

Preparation of Bacterial Inoculum for Acinetobacter Biofilm Formation

Preparation of Bacterial Inoculum for Acinetobacter Biofilm Formation  

Beredning av bakteriell inokulat för bildning av Acinetobacter Biofilm

Assessing Acinetobacter Biofilm Viability Count

Assessing Acinetobacter Biofilm Viability Count  

Bedömning av antalet livsduglighetsfaktorer för acinetobacterbiofilm

Acinetobacter Biofilm Visualization by Confocal Laser Scanning Microscopy (CLSM)

Acinetobacter Biofilmvisualisering med konfokal laserskanningsmikroskopi (CLSM)