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.

1. Preparation of bacterial inoculum
<ol>
	<li>Remove the glycerol stock vial stored at -80 &#176;C.</li>
	<li>Remove the bacterial strain (2-10 &#181;L) 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.</li>
	<li>Inoculate a commercially available blood agar plate (tryptic soy agar supplemented with 5% sheep blood, see Table of Materials) with the bacteria. 
	NOTE: Other media, such as nutrient agar or MacConkey agar, can also be used.</li>
	<li>Streak the agar surface using a sterile loop with intermittent flaming to thin it out.</li>
	<li>Place the agar plate upside down in an incubator and incubate at 30 &#176;C overnight for 20-24 h.</li>
	<li>Pick a single colony of the bacterial culture with a sterile needle and inoculate 5 mL sterile brain heart infusion broth (BHI, see Table of Materials) with the culture.</li>
	<li>Incubate the inoculated broth in a shaking incubator at 30 &#176;C overnight for 20-24 h at around 150 rpm.</li>
	<li>Transfer 1 mL of the overnight culture to a sterile microcentrifuge tube and centrifuge the overnight culture at 6,000 &#215; g for 10 min at room temperature (RT).</li>
	<li>Remove the supernatant with a pipette.</li>
	<li>Using a vortex, resuspend the pellet in 1 mL sterile phosphate-buffered saline (PBS).</li>
	<li>Centrifuge at 6,000 &#215; g for 10 min at RT and remove the supernatant.</li>
	<li>Resuspend the pellet in sterile ten-fold diluted BHI using vortex to an optical density of around 0.1 at 600 nm. 
	NOTE: The optical density of around 0.1 is equivalent to approximately 107 CFU/mL.</li>
</ol>
2. Biofilm quantification using crystal violet
<ol>
	<li>Add 200 &#181;L inoculum to each well of a sterile polystyrene 96-well plate (see Table of Materials) and add 200 &#181;L deionized water to each of the outermost wells to prevent the inner wells from drying out15.</li>
	<li>Add 200 &#181;L ten-fold diluted BHI to each well of the same plate as a negative control.</li>
	<li>Incubate the plate at 25 &#176;C for 24 h.</li>
	<li>Remove the supernatant with a pipette. 
	NOTE: Multichannel pipette is often more convenient for multiple samples.</li>
	<li>Carefully add 300 &#181;L PBS to each well and remove the supernatant with a pipette. 
	&#8203;NOTE: Multichannel pipette is often more convenient for multiple samples.</li>
	<li>Repeat step 2.5 two more times.</li>
	<li>Add 200 &#181;L crystal violet solution (1%) (see Table of Materials) to each well and incubate at RT for 30 min.</li>
	<li>Repeat steps 2.4-2.6.</li>
	<li>Add 200 &#181;L of absolute ethanol to each well and incubate for 15 min at RT. Mix thoroughly by pipetting up and down several times.</li>
	<li>Transfer 100 &#181;L elution from each well to a new 96-well plate.</li>
	<li>Measure the absorbance at 595 nm using a microplate reader (see Table of Materials).</li>
	<li>When the absorbance exceeds 2.0, make a proper dilution of the sample to the absorbance below 2.0 in absolute ethanol and measure it as in step 2.11. Then, calculate the absorbance of the sample (final value) by multiplying the observed value by the dilution factor.</li>
	<li>Calculate the true absorbance of the samples by subtracting the average value of the negative control from the value of each well of the test samples on the same well plate.</li>
</ol>
3. Biofilm viability count
<ol>
	<li>Add 1 mL inoculum (step 1) to each well of a sterile polystyrene 12-well plate15. Incubate the plate at 25 &#176;C for 24 h. Remove the supernatant with a pipette.</li>
	<li>Carefully add 1.5 mL sterile PBS to each well and remove the supernatant with a pipette. Add 1 mL sterile PBS to each well.</li>
	<li>Scrape off the bottom and wall surfaces of the well using fitted cell scrapers.</li>
	<li>Transfer the harvested cell suspension to a sterile plastic tube (10-1) containing 9 mL saline (0.85% NaCl) and 10-20 glass beads (see Table of Materials). 
	NOTE: To collect any residual biofilm debris left on the bottom surface of the well, it can be resuspended and transferred by pipette using saline from the 10-1 tube.</li>
	<li>Vortex the 10-1 tube for 60 s at a maximum speed.</li>
	<li>Make a 10-fold serial dilution by transferring 1 mL to the next sterile tube (10-2) containing 9 mL saline (0.85 % NaCl) up to 10-7.</li>
	<li>Spread 100 &#181;L from each dilution on Acinetobacter agar (see Table of Materials) and incubate the agar plates at 30 &#176;C for 24-42 h.</li>
	<li>Count the number of the typical red colonies on each plate manually in the range between 10 and 250.</li>
	<li>Calculate the number of viable cells in the biofilm of each well by using the equation below: 
	Viable cells = Number of colonies&#160;&#215; Dilution factors&#160;&#215; 10</li>
</ol>
4. Biofilm visualization using confocal laser scanning microscopy (CLSM)
<ol>
	<li>Add 200 &#181;L inoculum to each well of a sterile 96-well plate intended for microscopic analysis.</li>
	<li>Incubate the plate at 25 &#176;C for 24 h.</li>
	<li>Remove the supernatant with a pipette.</li>
	<li>Carefully add 300 &#181;L filter-sterilized deionized water to each well and remove the supernatant with a pipette.</li>
	<li>Repeat step 4.4.</li>
	<li>Prepare the mixture of SYTO 9 and propidium iodide (see Table of Materials) diluted in filter-sterilized deionized water to a final concentration of 10 &#181;M and 60 &#181;M, respectively.</li>
	<li>Add the mixture to each well at 200 &#181;L and incubate the plate for 20-30 min at RT in the dark.</li>
	<li>Repeat steps 4.3-4.4</li>
	<li>Visualize the bottom-surface attached biofilms using CLSM with the excitation wavelength of 488 nm and 561 nm and the emission wavelength range of 499-535 nm and 568-712 nm, respectively.</li>
</ol>

Assessment of &lt;em&gt;Acinetobacter&lt;/em&gt; Biofilm Formation and Characterization of Biofilms

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The authors have nothing to disclose.

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 &#176;C for the growth and 25 &#176;C for the biofilm formation of Acinetobacter. 30 &#176;C was used because many stu...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well cell culture plate</td>
			<td>SPL</td>
			<td>30096</td>
			<td>Polystyrene 96-well plate</td>
		</tr>
		<tr>
			<td>BHI (Brain Heart Infusion) broth</td>
			<td>Merck KGaA</td>
			<td>1.10493.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar Base Plate</td>
			<td>KisanBio</td>
			<td>MB-B1005-P50</td>
			<td>Growth media for Acinetobacter</td>
		</tr>
		<tr>
			<td>CHROMagar Acinetobacter</td>
			<td>CHROMagar</td>
			<td>AC092</td>
			<td>Selective plate for Acinetobacter</td>
		</tr>
		<tr>
			<td>Crystal violet solution</td>
			<td>Sigma-Aldrich</td>
			<td>V5265</td>
			<td></td>
		</tr>
		<tr>
			<td>Filmtracer LIVE/DEAD biofilm viability kit</td>
			<td>Invitrogen</td>
			<td>L10316</td>
			<td>SYTO9 and propidium iodide</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200 PRO NanoQuant</td>
			<td>Biofilm measurement</td>
		</tr>
		<tr>
			<td>RBC Glass Plating Beads</td>
			<td>RBC</td>
			<td>RG001</td>
			<td>Glass beads</td>
		</tr>
		<tr>
			<td>&#956;-Plate 96 Well Black</td>
			<td>ibidi</td>
			<td>89621</td>
			<td>Microplate intended for CLSM</td>
		</tr>
	</tbody>
</table>

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.

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.

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 Stepanovi&#263; et al.16, all...

Watch this Scientific Journal Video about A Comprehensive Protocol for Acinetobacter Biofilm Quantification, Assessment, and Visualization at JoVE.com

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, ...

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1.4K Views.  Korea Food Research Institute. 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 vi...

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

Preparation of Bacterial Inoculum for Acinetobacter Biofilm Formation

Preparation of Bacterial Inoculum for Acinetobacter Biofilm Formation  

To begin the process of bacterial inoculum preparation, collect 2 to 10 microliters of the bacterial strain from the vial, using a sterile pipette tip, and inoculate a commercially-available blood agar plate with it. Using a sterile loop, streak the agar surface, thinning it out with intermittent flaming. Incubate the prepared agar plate in an upside-down position at 30 degrees Celsius for about 20 to 24 hours.Next, select a single colony of the bacterial culture with a sterile needle and inoculate five milliliters of sterile BHI broth with this culture. Incubate the inoculated broth in a shaking incubator at 30 degrees Celsius at a speed of 150 rotations per minute for about 20 to 24 hours. Then transfer one milliliter of the culture to a sterile micro-centrifuge tube and centrifuge at 6, 000 g and room temperature for 10 minutes.Following centrifugation, discard the supernatant. Add one milliliter of sterile phosphate-buffered saline, or PBS, to the pellet, and re-suspend it using a vortex. Centrifuge the re-suspended pellet at 6, 000 g for 10 minutes at room temperature and discard the supernatant.This time re-suspend the resulting pellet in sterile, tenfold-diluted BHI broth using vortex. Ensure to achieve an optical density of around 0.1 at 600 nanometers.

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.

Assessing Acinetobacter Biofilm Viability Count

Assessing Acinetobacter Biofilm Viability Count  

Begin by adding one milliliter of the bacterial inoculum to each well of a sterile polystyrene 12 well plate. Incubate the plate at 25 degrees Celsius for 24 hours. Then, remove the supernatant using a pipette.Carefully add 1.5 milliliters of sterile phosphate buffered saline or PBS to each well, and again, remove the supernatant with the pipette. After adding one milliliter of sterile PBS to each well once more, use fitted cell scrapers to scrape off the bottom and wall surfaces of the wells. Transfer the harvested cell suspension to a sterile plastic tube marked as 10 to the negative one containing nine milliliters of saline and 10 to 20 glass beads.Vortex the tube for 60 seconds at maximum speed. Next, perform a tenfold serial dilution by transferring one milliliter of the cell suspension from the previous tube to another sterile tube marked 10 to the negative two containing nine milliliters of saline solution. Continue this process of serial dilution up to tube 10 to the negative seven.Spread 100 microliters from each dilution onto separate acinetobacter agar plates. Incubate the agar plates at 30 degrees Celsius for 24 to 42 hours. Then count the number of typical red colonies on each plate manually, considering those within the range between 10 and 250.All the acinetobacter isolates except for A.bouvetii had cells equivalent to seven to eight log CFU per well in their biofilms. While A.bouvetii had a much lower cell number at 4.4 Log CFU.

Acinetobacter Biofilm Visualization by Confocal Laser Scanning Microscopy (CLSM)

Begin by adding 200 microliters of bacterial inoculum into each well of a sterile 96-well plate prepared for microscopic analysis. Incubate the plate at 25 degrees Celsius for 24 hours before removing the supernatant with the pipette. Next, carefully add 300 microliters of filter-sterilized deionized water into each well.Remove the supernatant using a pipette once again. In another tube, prepare a diluted mixture of SYTO 9 and propidium iodide and filter-sterilized deionized water to the respective desired final concentrations. Then add 200 microliters of the mixture into each well and incubate the plate for 20 to 30 minutes at room temperature in a dark setting.Confocal laser scanning microscopy revealed that biofilm formation significantly varied depending on the strains. A substantial amount of biofilm was found in Acinetobacter junii, Acinetobacter baumannii, and Acinetobacter ursingii with distinct biofilm morphologies.