acinetobacter biofilm quantification using crystal violet

아시네토박터 생물막 형성 평가 및 생물막의 특성화

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.

저자 스포트라이트: Acinetobacter Biofilm 정량화, 평가 및 시각화를 위한 포괄적인 프로토콜 

Acinetobacter Biofilms에 대한 정량화, 생존력 평가 및 시각화 전략

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  

아시네토박터 Crystal Violet을 사용한 생물막 정량

시작하려면 멸균 폴리스티렌 200웰 플레이트의 각 웰에 96마이크로리터의 박테리아 접종물을 추가합니다. 그런 다음 가장 바깥쪽 우물에 200마이크로리터의 탈이온수를 추가하여 내부 우물이 마르지 않도록 합니다. 섭씨 25도에서 24시간 동안 96웰 플레이트를 배양합니다.배양 후 피펫을 사용하여 각 웰의 상층액을 버립니다. 각 웰에 300마이크로리터의 PBS를 조심스럽게 추가하고 피펫을 사용하여 PBS를 다시 제거합니다. 200 마이크로 리터의 1 % 크리스탈 바이올렛 용액을 각 웰에 첨가한 후 플레이트를 실온에서 30 분 동안 배양합니다.그런 다음 각 웰에 200마이크로리터의 절대 에탄올을 분배한 후 플레이트를 실온에서 15분 동안 그대로 둡니다. 각 웰의 내용물을 위아래로 반복적으로 피펫팅하여 철저한 혼합을 보장합니다. 마이크로플레이트 리더를 사용하여 595나노미터에서 웰의 흡광도를 측정하여 생물막 질량을 측정합니다.biofilms의 수는 A.radioresistens가 약한 biofilm를 형성하는 그러나, A.bouvetii를 제외하고, A.bouvetii를 제외하고, 분리된 Acetobacter 전부는, biofilms를, 형성하는 그러나, 0.04에서 1.69에 광학적인 조밀도 또는 OD 가치에서 배열하는 긴장에 따라서 매우 변화했습니다. A.junii와 A.baumannii는 온건한 biofilms를 형성하고, A.pittii와 A.ursingii는 강한 biofilms를 형성했습니다.

acinetobacter-biofilm-quantification-using-crystal-violet

Research

JoVE Journal

Biology

Acinetobacter Biofilm Quantification Using Crystal Violet

520 조회수.  Korea Food Research Institute. 시작하려면 멸균 폴리스티렌 200웰 플레이트의 각 웰에 96마이크로리터의 박테리아 접종물을 추가합니다. 그런 다음 가장 바깥쪽 우물에 200마이크로리터의 탈이온수를 추가하여 내부 우물이 마르지 않도록 합니다. 섭씨 25도에서 24시간 동안 96웰 플레이트를 배양합니다.배양 후 피펫을 사용하여 각 웰의 상층액을 버립니다. 각 웰에 300마이크로리터의 PBS를 조심스럽게 추...

비디오: 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, ...

연구

생물학

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.

아시네토박터 생물막 형성을 위한 박테리아 접종물의 제조

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.

아시네토박터 생물막 생존력 계수 평가

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.