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

Assessment of Acinetobacter Biofilm Formation and Characterization of Biofilms

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.

Author Spotlight: A Comprehensive Protocol for Acinetobacter Biofilm Quantification, Assessment, and Visualization 

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

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 Biofilm Quantification Using Crystal Violet

To begin, add 200 microliters of the bacterial inoculum to each well of a sterile polystyrene 96-well plate. Then add 200 microliters of deionized water into the outermost wells to keep the inner wells from drying out. Incubate the 96-well plate at 25 degrees Celsius for 24 hours.Following incubation, discard the supernatant in each well using a pipette. Carefully add 300 microliters of PBS to each well and remove the PBS again by using the pipette. Add 200 microliters of 1%crystal violet solution to each well before incubating the plate at room temperature for 30 minutes.Then after dispensing 200 microliters of absolute ethanol in each well, leave the plate for 15 minutes at room temperature. Ensure thorough mixing by pipetting the contents in each well up and down repeatedly. Using a microplate reader, measure the absorbance of the wells at 595 nanometers to determine the biofilm mass.The number of biofilms varied greatly depending on the strains, ranging from an optical density or OD value of 0.04 to 1.69 All of the Acetobacter isolates, except for A.bouvetii, formed biofilms, while A.radioresistens formed a weak biofilm. A.junii and A.baumannii formed moderate biofilms, and A.pittii and A.ursingii formed strong biofilms.

Research

JoVE Journal

Biology

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