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Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

Published: August 4th, 2023



1Korea Food Research Institute, 2Department of Food Biotechnology, Korea University of Science and Technology, 3Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute

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, 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 °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.

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 Acinet....

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1. Preparation of bacterial inoculum

  1. Remove the glycerol stock vial stored at -80 °C.
  2. Remove the bacterial strain (2-10 µL) from the vial using a sterile pipette tip.
    NOTE: Acinetobacter strains, A. bouvetii (13-1-1), A. junii (13-1-2), A. pittii (13-2-5), A. baumannii (13-2-9), A. radioresistens (20-1), and A. ursingii (24-1) are used in this protocol.
  3. Inoculate a commercially available blood .......

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Following the protocol, the biofilms of Acinetobacter isolates, originally isolated from kitchen surfaces, were formed on a polystyrene 96-well plate, stained with crystal violet, and the dyes were solubilized in ethanol and measured for biofilm mass (Figure 1). The number of biofilms greatly varied depending on the strains ranging from OD 0.04 to 1.69 (Figure 1). Based on the criteria established by Stepanović et al.16, all.......

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Using the protocol described, the biofilm formation of Acinetobacter isolates with varying degrees was measured, visualized, and the viable cells in the biofilms were estimated (Figure 1, Figure 2, and Figure 3).

In this protocol, two different temperatures were used, 30 °C for the growth and 25 °C for the biofilm formation of Acinetobacter. 30 °C was used because many stu.......

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This research was supported by the Main Research Program (E0210702-03) of the Korea Food Research Institute (KFRI), funded by the Ministry of Science and ICT.


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NameCompanyCatalog NumberComments
96-well cell culture plateSPL30096Polystyrene 96-well plate
BHI (Brain Heart Infusion) brothMerck KGaA1.10493.0500
Blood Agar Base PlateKisanBioMB-B1005-P50Growth media for Acinetobacter
CHROMagar AcinetobacterCHROMagarAC092Selective plate for Acinetobacter
Crystal violet solutionSigma-AldrichV5265
Filmtracer LIVE/DEAD biofilm viability kitInvitrogenL10316SYTO9 and propidium iodide
Microplate readerTecanInfinite M200 PRO NanoQuantBiofilm measurement
RBC Glass Plating BeadsRBCRG001Glass beads
μ-Plate 96 Well Blackibidi89621Microplate intended for CLSM

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