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Jan 2020

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Candida Biofilm Formation Assay on Essential Oil Coated Silicone Rubber
精油包覆硅橡胶上念珠菌生物膜形成的实验研究   

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Abstract

Development of biofilm associated candidemia for patients with implanted biomaterials causes an urgency to develop antimicrobial and biofilm inhibitive coatings in the management of recalcitrant Candida infections. Recently, there is an increase in the number of patients with biofilm formation and resistance to antifungal therapy. Therefore, there is a growing interest to use essential oils as coating agents in order to prevent biomaterial-associated Candida infections. Often high costs, complicated and laborious technologies are used for both applying the coating and determination of the antibiofilm effects hampering a rapid screening of essential oils. In order to determine biofilm formation of Candida on essential oil coated surfaces easier, cheaper and faster, we developed an essential oil (lemongrass oil) coated surface (silicone-rubber) by using a hypromellose ointment/essential oil mixture. Furthermore, we modified the “crystal violet binding assay” to quantify the biofilm mass of Candida biofilm formed on the lemongrass oil coated silicone rubber surface. The essential oil coating and the biomass determination of biofilms on silicone rubber can be easily applied with simple and accessible equipment, and will therefore provide rapid information about whether or not a particular essential oil is antiseptic, also when it is used as a coating agent.

Keywords: Biofilm formation (生物膜形成), Biomaterial infections (生物材料的感染), Candida (念珠菌), Candidemia (念珠菌血症), Essential oils (香精油), Essential oil coated surfaces (精油涂层表面)

Background

In recent years, the frequency of Candida infections caused by both nosocomial and opportunistic Candida strains has increased rapidly especially due to the growing number of transplant recipients, cancer patients and patients who receive immunosuppressive therapy (Sanguinetti et al., 2015). Apart from this, development of candidemia in patients with implanted biomaterials such as catheters (Simitsopoulou et al., 2014), dental implants (Li et al., 2012), voice prostheses (Talpaert et al., 2015) and prosthetic devices (Ramage et al., 2006) causes an urgency to develop preventive strategies such as antimicrobial coatings in the management of recalcitrant Candida infections (Ramage et al., 2006). Since biofilm formation plays a pivotal role in the development of recalcitrant candidemia (Simitsopoulou et al., 2014), not only antimicrobial therapies, but also antibiofilm strategies are needed for the prevention of biomaterial-associated Candida infections. Recently, a growing increase in the incidence of candidemia has been associated with the increase in biofilm formation and resistance to antifungal therapy (Francisconi et al., 2020). Substances obtained from traditional medicinal plants, such as volatile essential oils have been regarded as attractive sources for new antimicrobials(Francisconi et al., 2020). Essential oils, originating from different parts of a variety of aromatic plants not only have antimicrobial but also have antibiofilm effects against various microorganisms (Akthar et al., 2014; Kim et al., 2016; Sahal et al., 2019 and 2020). Therefore, the antibiofilm effects of several essential oils against different Candida species have been studied for years (Almeida et al., 2016; Kryvtsova et al., 2019). Recently, essential oils have also been evaluated as agents that prevent biomaterial-associated Candida infections. In particular, there is a growing interest in the use of a lemongrass essential oil as a coating agent (Sahal et al., 2019 and 2020). Often high costs and complicated technologies are used for both applying the coating and determination of the antibiofilm effects. For example, Liakos et al. (2017) used electrospinning to apply a cellulose acetate based coating containing the essential oil and Anghel et al. (2012) used Confocal Laser Scanning Microscopy images to asses fungal biofilm architecture on Fe3O4/C18/essential oil coated ProvoxTM voice prostheses. These techniques are often very expensive, laborious and require highly qualified equipment and personnel. Thus, they constrain an ease and rapid screening of essential oils. Therefore, we aimed to develop cheap, effective and easily applicable protocols to rapidly screen the anti-candida biofilm effects of various essential oils coated on silicone rubber.


In the first part, we describe the preparation of essential oil coated silicone rubber surface using a hypromellose ointment/essential oil mixture. In this part, hypromellose ointment was used as a vehicle because it mixes well with essential oils and because of its good adhesive properties to wetted surfaces. In the second part, we describe the determination of the biofilm mass of Candida biofilm on these surfaces using a modified crystal violet binding assay. Both protocols can be applied easily and rapidly with simple and accessible equipment.

Materials and Reagents

  1. VST Silicone elastomer (low viscosity, translucent silicone) (10 × 10 × 1.5 mm sheets) (Polymerization conditions: 5-bar pressure, 45 °C, 90 min.) (Maxillofacial Silicone System, Technovent Ltd., South Wales, UK, catalog number: VST-50HD Shore A 38, cure time overnight).

  2. Petri dish (90 mm × 17 mm) (Isolab, catalog number: 0 8102061 )

  3. Erlenmayer Flask (250 ml) (Isolab, catalog number: 027.01.250 )

  4. 6-Well plates (Biofil, catalog number: TCP 011006 )

  5. 24-Well plates (Biofil, catalog number: TCP 011024 )

  6. Centrifugal tube 50 ml (Orange Scientific, catalog number: 4440100N )

  7. Candida strains (Clinical Candida isolates, previously characterized as highly biofilm forming and identified using the 18S Ribosomal RNA Gene Sequence Analysis; or biofilm-positive ATCC Candida strains such as Candida albicans (Robin) Berkhout (ATCC®, catalog number: MYA-274 MYA-274TM )

  8. Lemongrass essential oil (Merck, catalog number: 0 5521501 )

  9. Hypromellose ointment [Hypromellose 4000 mPa.s 20% (w/w); Soft paraffin white 80% (w/w)], [FNA (Formularium der Nederlandse Apothekers), Dutch Pharmacists' Formulary, 2013], (Fagron, catalog number: 102064 )

  10. Crystal Violet Powder (Merck, catalog number: C0775-100G )

  11. Brain Heart Infusion (BHI) Powder (Merck, catalog number: M110493.0500 )

  12. Agar-agar (Merck, catalog number: M101613.0500 )

  13. Ethanol 96% (v/v) (Isolab, catalog number: 9200262500 )

  14. Extran MA 02 (Merck, catalog number: 107553 )

  15. K2HPO4 (Merck, catalog number: 7758-11-4)

  16. KH2PO4 (Merck, catalog number: 7778-77-0 )

  17. Distilled Water

  18. Brain Heart Infusion Broth (see Recipes)

  19. Brain Heart Infusion Agar (see Recipes)

  20. 1% Crystal Violet Solution (see Recipes)

  21. 10 mM Potassium phosphate buffer (see Recipes)

Equipment

  1. Bunsen burner

  2. Porcelain mortar [Outside diameter 10.5 cm, inner diameter 9.0 cm, inner depth 4.5 cm. Content 100 ml. Glazed to grinding surface, grinding surface unglazed and porcelain pestle (Length 11 cm, unglazed on grinding surface, further glazed)] (Haldenwanger, Berlin)

  3. Forceps

  4. Spectrophotometer (UV-Visible Spectrophotometer; Shimadzu, model: UV-1700, catalog number: 206-98366A )

  5. Centrifuge (Eppendorf, model: 5810R, with an Eppendorf Swing-bucket rotor A-4-62 , catalog number: 5810000327)

  6. pH Meter (ConsortTM C3010 Benchtop Multiparameter Analyzer) (Fisher Scientific, catalog number: 11772009 )

  7. Thoma Counting Chamber (Neubauer, catalog number: C964110 )

  8. Autoclave (Priorclave, Catalog number: 12758935 )

  9. Light Microscope (LEICA, model: DM500 )

  10. Incubator (Static) (Dedeoglu, catalog number: MMF-KM-068 )

Software

  1. MINITAB (Version 18)

  2. ImageJ (Version 1.49)

Procedure

  1. Preparation of essential oil coated silicone rubber surfaces by using hypromellose ointment (see also Figure 1)

    1. Mix hypromellose ointment with a particular essential oil (w/w) under aseptic conditions using a sterile porcelain mortar and pestle.

    2. Place a layer of essential oil/hypromellose ointment mixture (50 mg) (w/w) in a sterile Petri dish under sterile conditions by using a Bunsen burner.

    3. Clean silicone rubber surface sheets by rinsing them with 2% Extran MA02 in water followed by tap water, 70% ethanol solution (v/v) and sterile demineralized water.

    4. Place the sterile silicone rubber surface sheet (10 × 10 mm) on top of a layer of essential oil/hypromellose ointment mixture (w/w).

    5. Apply a gentle pressure onto the silicone rubber surface sheet to let the essential oil/hypromellose ointment mixture stick onto the material.

    6. Take essential oil coated silicone rubber surface sheet gently out of the Petri dish by using sterile forceps and place it in a well of a sterile 6-Well plate for the further analysis.

    7. Place the essential oil coated silicone rubber surface sheet vertical on a sterile Petri dish and put them under a light microscope (×10) in order to capture an image that contains thicknesses of both the silicone rubber sheet and the coating.

    8. Set the thickness of the silicone rubber sheet as 1.5 mm and estimate the thickness of the essential oil/hypromellose ointment coatings on the silicone rubber surfaces by using ImageJ Software.



      Figure 1. Preparation of essential oil coated silicone rubber by using essential oil/hypromellose ointment mixture. A. Placement of essential oil/hypromellose ointment mixture in a sterile Petri dish. B. Placement of a sterile biomaterial surface sheet on top of a layer of essential oil/hypromellose ointment mixture and application of a gentle pressure onto the biomaterial surface sheet to let the essential oil/hypromellose ointment mixture stick onto the material. C. Taking essential oil coated surface sheet gently out of the Petri dish.


  2. Determination of Candida biofilm formation on essential oil coated surfaces (see also Figure 2)

    1. Inoculate Candida strains from stock culture (grown in BHI with glycerol) into Brain Heart Infusion (BHI) agar by a streak plating technique and incubate them for 24 h at 37 °C under aerobic conditions.

    2. Inoculate single colonies of Candida strains for pre-culture preparations into 10 ml BHI broth and incubate overnight at 37 °C without shaking.

    3. Inoculate 10 ml preculture into the 200 ml BHI broth and grow it for 24 h at 37 °C without shaking to obtain main culture.

    4. Harvest Candida cells by centrifugation at 3,220 × g for 10 min at 5 °C and wash 3 times with 30 ml 10 mM potassium phosphate buffer (pH 7.0).

    5. Count Candida cells that exist in 8 different medium squares of a Thoma Counting Chamber under a light microscope with 40× objective. Calculate the average of the cells for a single medium square and calculate the Candida cell density considering the volume of a single medium square of the Thoma Counting Chamber. Adjust cell density to 3 × 106 cells/ml in BHI broth.

    6. Place the essential oil coated silicone rubber surfaces, silicone rubber surfaces with only hypromellose ointment and the silicone rubber surfaces without any coating (positive controls) into the wells of a 24-well plate.

    7. Add 2 ml of Candida suspensions to the wells of 24-well plates that include the different silicone rubber surfaces and incubate them for 7 days at 37 °C under aerobic conditions without shaking.

    8. After 7 days of incubation, wash gently three times the materials harboring biofilm with 10 mM potassium phosphate buffer (pH 7) and stain them with a 1% (w/v) solution of crystal violet in sterile distilled water for 20 min at 25 °C.

    9. Subsequently, gently rinse the materials with 10 mM potassium phosphate buffer (pH 7.0) again until no crystal violet residue left in the washing solution.

    10. Dissolve crystal violet bound to the biofilm on the different tested surfaces in 2 ml ethanol (96%; v/v) for 20 min (O’Toole, 2011).

    11. Measure the absorbance of dissolved crystal violet at 560 nm using a spectrophotometer (O’Toole, 2011).



      Figure 2. Biofilm formation of Candida. Images of A) silicone rubber surface B) Biofilm formation on 8% Lemongrass oil coated silicone rubber surface C) Biofilm formation on 0% Lemongrass oil coated silicone rubber surface (hypromellose ointment alone) D) Biofilm formation on silicone rubber without coating (positive control).

Data analysis

  1. Set the absorbance value of the silicone rubber surface without any coating and microorganism, but following the same procedure as the biofilm growth and crystal violet staining, as a negative control. Subtract this value from the absorbance values of the surfaces with biofilms.

  2. Evaluate biofilm formation on the silicone rubber surface without coating as a positive control and set it as 100%. Set silicone rubber surface coated with hypromellose ointment without any essential oil as 0% essential oil coated surface.

  3. Measure the absorbance values of biofilm formations of treatments and calculate decrease (%) in biofilm formation of treatments relative to the positive control, using the equation:

    % Decrease = [(Absorbance Positive Control (560 nm) – Absorbance Treatment (560 nm))/Absorbance Positive Control (560 nm)] × 100%

  4. Perform statistical analysis using MINITAB 18 software.

  5. Apply Anderson-Darling test to determine if biofilm formation and biofilm inhibition data were normally distributed. Following this, apply Levene's test to assess homogeneity of variances.

  6. In case the results are found to be normally distributed, apply two factor experimental design and do pairwise comparisons by the Tukey test. In case the results are not found as normally distributed, use non-parametric tests (Kruskal-Wallis and Mann-Whitney U tests) to evaluate the results.

Notes

  1. Determination of biofilm formation of Candida on essential oil coated silicone rubber surfaces by modified crystal violet binding assay need to be performed at least in triplicate, including controls.

  2. When the absorbance is too high, dilute the dissolved crystal violet and multiply the absorbance value by the dilution factor.

Recipes

  1. Brain Heart Infusion Broth (1 L)

    Dissolve 37 g BHI powder in 1 L distilled water and sterilize the medium at 121 °C for 15 min using an autoclave.

  2. Brain Heart Infusion Agar (1 L)

    Dissolve 37 g BHI powder and 30 g Agar-Agar powder in 1 L distilled water and place the media into sterile Petri dishes after sterilization at 121 °C for 15 min using an autoclave.

  3. 1% Crystal Violet Solution (w/v)

    Dissolve 5 g crystal violet in 500 ml distilled water and incubate the crystal violet solution (1%, w/v) in shaking incubator for 2 h at 150 rpm, at room temperature.

  4. 10 mM Potassium Phosphate Buffer (pH 7.0) (1 L)

    1. Dissolve 4.35 g K2HPO4 and 3.4 g KH2PO4 in 100 ml distilled water to obtain stock 0.5 M Potassium Phosphate Buffer.

    2. Dilute 0.5 M potassium phosphate buffer to 10 mM potassium phosphate buffer.

    3. Adjust the pH of the buffer to 7.0 by using pH meter and sterilize the buffer at 121 °C for 15 min using an autoclave.

Acknowledgments

This study was supported by funding received from Scientific Research Projects Coordination Unit (grant number: FDK-2016-10821) of Hacettepe University, Ankara, Turkey. This protocol was derived from Sahal et al. (2020).

Competing interests

The authors declare no competing financial interests.

References

  1. Akthar, M. S., Degaga, B. and Azam, T. (2014). Antimicrobial activity of essential oils extracted from medicinal plants against the pathogenic microorganisms : A review. Biol Sci Pharm Res 2(1): 1-7.
  2. Almeida, Lde, F., Paula, J. F., Almeida, R. V., Williams, D. W., Hebling, J. and Cavalcanti, Y. W. (2016). Efficacy of citronella and cinnamon essential oils on Candida albicans biofilms. Acta Odontol Scand 74(5): 393-398.
  3. Anghel, I., Grumezescu, V., Andronescu E., Anghel G. A. Grumezescu, A. M., Mihaiescu, D. E. and Chifiriuc M. C. (2012). Protective effect of magnetite nanoparticle/Salvia officinalis essential oil hybrid nanobiosystem against fungal colonization on the Provox® voice section prosthesis. Dig J Nanomater Biostructures 7(3): 1205-1212.
  4. Francisconi, R. S., Huacho, P. M. M., Tonon, C. C., Bordini, E. A. F., Correia, M. F., Sardi, J. C. O. and Spolidorio, D. M. P. (2020). Antibiofilm efficacy of tea tree oil and of its main component terpinen-4-ol against Candida albicans. Braz Oral Res 34: e050.
  5. Kim, Y. G., Lee, J. H., Gwon, G., Kim, S. I., Park, J. G. and Lee, J. (2016). Essential Oils and Eugenols Inhibit Biofilm Formation and the Virulence of Escherichia coli O157:H7.Sci Rep 6: 36377.
  6. Kryvtsova, M. V., Salamon, I., Koscova, J., Bucko, D. and Spivak, M. (2019). Antimicrobial, antibiofilm and biochemichal properties of Thymus vulgaris essential oil against clinical isolates of opportunistic infections. Biosyst Divers 27(3): 270-275.
  7. Li, J., Hirota, K., Goto, T., Yumoto, H., Miyake, Y. and Ichikawa, T. (2012). Biofilm formation of Candida albicans on implant overdenture materials and its removal. J Dent 40(8): 686-692.
  8. Liakos, I. L., Holban, A. M., Carzino, R., Lauciello, S. and Grumezescu, A. M. (2017). Electrospun Fiber Pads of Cellulose Acetate and Essential Oils with Antimicrobial Activity. Nanomaterials (Basel) 7(4): 84.
  9. O'Toole, G. A. (2011). Microtiter dish biofilm formation assay. J Vis Exp 47: 2437.
  10. Ramage, G., Martinez, J. P. and Lopez-Ribot, J. L. (2006). Candida biofilms on implanted biomaterials: a clinically significant problem. FEMS Yeast Res 6(7): 979-986.
  11. Sahal, G., Woerdenbag, H. J., Hinrichs, W. L. J., Visser, A., Tepper, P. G., Quax, W. J., van der Mei, H. C. and Bilkay, I. S. (2020). Antifungal and biofilm inhibitory effect of Cymbopogon citratus (lemongrass) essential oil on biofilm forming by Candida tropicalis isolates; an in vitro study. J Ethnopharmacol 246: 112188.
  12. Sahal, G., Nasseri, B., Ebrahimi, A. and Bilkay, I. S. (2019). Electrospun essential oil-polycaprolactone nanofibers as antibiofilm surfaces against clinical Candidatropicalis isolates. Biotechnol Lett 41(4-5): 511-522.
  13. Sanguinetti, M., Posteraro, B. and Lass-Florl, C. (2015). Antifungal drug resistance among Candida species: mechanisms and clinical impact. Mycoses 58 Suppl 2: 2-13.
  14. Simitsopoulou, M., Kyrpitzi, D., Velegraki, A., Walsh, T. J. and Roilides, E. (2014). Caspofungin at catheter lock concentrations eradicates mature biofilms of Candida lusitaniae and Candida guilliermondii. Antimicrob Agents Chemother 58(8): 4953-4956.
  15. Talpaert, M. J., Balfour, A., Stevens, S., Baker, M., Muhlschlegel, F. A. and Gourlay, C. W. (2015). Candida biofilm formation on voice prostheses. J Med Microbiol 64(Pt 3): 199-208.

简介

[摘要]对于植入了生物材料的患者,与生物膜相关的念珠菌血症的发展导致迫切需要开发抗微生物和生物膜抑制性涂层,以应对顽固的念珠菌感染。近来,具有生物膜形成和抗真菌治疗抗性的患者数量增加。因此,有越来越多的兴趣使用精油作为涂层剂以防止生物材料-相关念珠菌感染。通常,高成本,复杂且费力的技术既用于涂覆涂层,又用于确定抗生物膜作用,这妨碍了对精油的快速筛选。为了更轻松,更便宜,更快速地确定精油涂层表面上念珠菌的生物膜形成,我们使用羟丙甲纤维素软膏/精油混合物开发了精油(柠檬皮油)涂层表面(硅橡胶)。此外,我们修改了“结晶紫结合法”以定量在柠檬草油涂层的硅橡胶表面上形成的念珠菌生物膜的生物膜质量。硅橡胶上的精油涂层和生物膜的生物量测定可通过简单且易于接近的设备轻松进行,因此,即使将特定的精油用作涂层剂,也将提供有关特定精油是否具有防腐性的快速信息。


[背景技术[ 0002 ]近年来,由医院和机会性念珠菌菌株引起的念珠菌感染的频率迅速增加,特别是由于移植接受者,癌症患者和接受免疫抑制疗法的患者的数量增加(Sanguinetti等人,2015)。除此之外,在植入生物材料的患者中出现念珠菌血症,例如导管(Simitsopoulou等人,2014),牙科植入物(Li等人,2012),语音假体(Talpaert等人,2015)和修复装置(Ramage)等人,2006)迫切需要发展预防策略,例如在顽固性念珠菌感染的管理中使用抗微生物涂层(Ramage等人,2006)。由于生物膜的形成在顽固性念珠菌血症的发展中起着关键作用(Simitsopoulou等人,2014),因此不仅需要抗菌药物治疗,而且还需要采取抗生物膜策略来预防与生物材料相关的念珠菌感染。近来,念珠菌血症的发生率的增长与生物膜形成的增加和对抗真菌疗法的抗性有关(Franciscoi等,2020)。从传统药用植物中获得的物质,例如挥发性香精油,已被认为是新型抗菌剂的诱人来源(Franciscoi等,2020)。源自多种芳香植物不同部位的精油不仅具有抗微生物作用,而且还具有针对多种微生物的抗生物膜作用(Akthar等人,2014; Kim等人,2016; Sahal等人,2019和2020 )。因此,已经研究了数种精油对不同的念珠菌物种的抗生物膜作用(Almeida等,2016;Kryvtsova等,2019)。近日,精油也被评估为防止剂的生物材料-相关念珠菌感染。特别地,对使用柠檬草精油作为包衣剂的兴趣日益增长(Sahal等人,2019和2020)。通常,高成本和复杂的技术被用于施加涂层和确定抗生物膜作用。例如,Liakos等。Anghel等人(2017)使用静电纺丝法涂覆了含有精油的醋酸纤维素基涂料。(2012年)使用共聚焦激光扫描显微镜图像评估Fe 3 O 4 / C 18 /精油包被的Provox TM语音假体上的真菌生物膜结构。这些技术通常非常昂贵,费力并且需要高素质的设备和人员。因此,它们限制了精油的容易和快速筛选。因此,我们旨在开发便宜,有效且易于应用的方案,以快速筛选涂在硅橡胶上的各种精油的抗念珠菌生物膜作用。

在第一部分中,我们描述了使用羟丙甲纤维素软膏/精油混合物制备精油涂层的硅橡胶表面的方法。在这一部分中,使用羟丙甲纤维素软膏作为媒介,因为它与香精油混合良好,并且对湿润的表面具有良好的粘合性能。在第二部分中,我们描述了使用改良的结晶紫结合测定法测定这些表面上念珠菌生物膜的生物膜质量。可以使用简单易用的设备轻松快速地应用这两种协议。

关键字:生物膜形成, 生物材料的感染, 念珠菌, 念珠菌血症, 香精油, 精油涂层表面



材料和试剂


1. VST硅酮弹性体(低粘度的,半透明的硅树脂)(10 × 10 × 1 。5枚2mm片材)(聚合条件:5巴压力,45℃,90分钟)(颌面硅氧烷体系,Technovent有限公司,南英国威尔士,目录号:VST-50HD Shore A 38,固化时间为一整夜。     

2.培养皿(90毫米× 1 7毫米)(Isolab,目录号:08102061)     

3.锥形烧瓶(250毫升)(Isolab,目录号:027.01.250)     

4. 6孔板(Biofil,目录号:TCP 011006)     

5. 24孔板(Biofil,目录号:TCP 011024)     

6. 50 ml离心管(Orange Scientific,目录号:4440100N)     

7.念珠菌菌株(临床念珠菌分离物,先前表征为高度生物膜形成和使用18S rRNA基因序列分析鉴定;或生物膜阳性ATCC念珠菌菌株如白色念珠菌(罗宾)Berkhout(ATCC ® ,Ç atalog号:MYA -274 TM )     

8.柠檬草精油(默克,目录号:05521501)     

9.羟丙甲纤维素软膏[羟丙甲纤维素4000 mPa.s 20%(w / w);软石蜡白80%(w / w)] ,[ FNA(Formularium der Nederlandse Apothekers),荷兰药剂师配方,2013年] ,(Fagron,目录号:102064)     

10.结晶紫粉末(默克,目录号:C0775-100G) 

11.脑心脏输注(BHI)散剂(默克,目录号:M110493.0500) 

12.琼脂(Merck,目录号:M101613.0500) 

13. 96%乙醇(v / v)(Isolab,目录号:9200262500) 

14. Extran MA 02(默克,目录号:107553) 

15. K 2 HPO 4 (默克,目录号:7758-11-4) 

16. KH 2 PO 4 (默克,目录号:7778-77-0) 

17.蒸馏水 

18.脑心脏灌注液(请参阅食谱) 

19.脑心浸液琼脂(请参阅食谱) 

20. 1%结晶紫溶液(请参阅食谱) 

21. 10 mM磷酸钾缓冲液(请参见食谱) 



设备


本生灯
瓷钵[外径10.5厘米,内径9.0厘米,内径4.5厘米。内容量100毫升。给打磨表面上釉,打磨表面不上釉,瓷杵(长度11厘米,打磨表面上不打磨,再上釉)] (Haldenwanger,柏林)
钳子
分光光度计(UV可见分光光度计;岛津制作所,型号:UV- 1700 ,Ç atalog号:206-98366A)
离心机(的Eppendorf,型号:5810R,用Eppendorf摇摆斗转子A-4-62,Ç atalog编号:5810000327)
pH计(康索特TM C3010台式多参数分析仪)(Fisher Scientific公司,Ç atalog号:11772009)
托马计数室(Neubauer,目录号:C964110)
高压灭菌器(Priorclave,货号:12758935)
光学显微镜(LEICA,型号:DM500)
培养箱(静态)(Dedeoglu,Ç atalog号:MMF-KM-068)


软件


MINITAB(版本18)
ImageJ(版本1.49)


程序


使用羟丙甲纤维素软膏制备精油涂层的硅橡胶表面(另请参见图1)
在无菌条件下,使用无菌瓷研钵和杵将羟丙甲纤维素软膏与特定精油(w / w)混合。
通过使用本生灯在无菌条件下,在无菌培养皿中放置一层精油/羟丙甲纤维素软膏混合物(50 mg)(w / w)。
用水冲洗2%Extran MA02,然后用自来水,70%乙醇溶液(v / v)和无菌软化水冲洗,以清洁硅橡胶表面片。
将无菌硅橡胶表面薄片(10 × 10 mm)放在一层精油/羟丙甲纤维素软膏混合物(w / w)的上面。
在硅橡胶表面上轻轻施加压力,以使精油/羟丙甲纤维素软膏混合物粘在材料上。
使用无菌镊子将精油涂层的硅橡胶表面薄片轻轻地从培养皿中取出,并将其放在无菌的6孔板的孔中进行进一步分析。
放置精油涂覆硅橡胶表面片材上的无菌垂直P ETRI盘,并把它们在光学显微镜(下× 10),以便捕获包含硅橡胶片和涂层两者的厚度的图像。
使用ImageJ Software将硅橡胶片的厚度设置为1.5 mm,并估计硅橡胶表面上的精油/羟丙甲纤维素软膏涂层的厚度。






图1.通过使用精油/羟丙甲纤维素软膏混合物制备精油包被的硅橡胶。A.将精油/羟丙甲纤维素软膏混合物置于无菌培养皿中。B.将无菌生物材料表面薄片放置在精油/羟丙甲纤维素软膏混合物层的顶部上,并在生物材料表面薄片上施加适度的压力以使精油/羟丙甲纤维素软膏混合物粘在材料上。C.轻轻地从培养皿中取出涂有精油的表面层。           



测定在精油涂层表面上念珠菌生物膜的形成(另见图2)
通过条纹电镀技术将来自原种培养物中的念珠菌菌株(在BHI中用甘油培养)接种到脑心浸液(BHI)琼脂中,并在有氧条件下于37°C孵育24 h。
将念珠菌菌株的单个菌落接种到10 ml BHI肉汤中进行预培养,然后在37°C孵育过夜而不摇动。
在200 ml BHI肉汤中接种10 ml预培养物,使其在37°C下生长24 h,而无需摇动以获得主要培养物。
通过在5°C下以3,220 × g离心10分钟收获念珠菌细胞,并用30 ml 10 mM磷酸钾缓冲液(pH 7.0)洗涤3次。
计数念珠菌中存在的托马计数室的8米不同的介质的正方形在光学显微镜用40下细胞×物镜。计算单个培养基正方形的细胞平均值,并考虑Thoma计数室单个培养基正方形的体积,计算念珠菌细胞密度。在BHI肉汤中将细胞密度调整为3×10 6个细胞/ ml。
将精油涂层的硅橡胶表面,仅带有羟丙甲脂软膏的硅橡胶表面和不含任何涂层的硅橡胶表面(阳性对照)置于24孔板的孔中。
将2 ml念珠菌悬浮液添加到24孔板的孔中,其中包括不同的硅橡胶表面,并在有氧条件下于37°C孵育7天,不要摇晃。
孵育7天后,用10 mM磷酸钾缓冲液(pH 7)轻轻洗涤三倍带有生物膜的材料,并在无菌蒸馏水中用1%(w / v)的结晶紫溶液在25°C下染色20分钟。 C。
随后,再次用10 mM磷酸钾缓冲液(pH 7.0)轻轻冲洗材料,直到洗涤液中没有结晶紫残留。
将与生物膜结合的结晶紫溶解在2 ml乙醇(96%; v / v)中的不同测试表面上20分钟(O'Toole,2011)。
使用分光光度计(O'Toole,2011)在560 nm下测量溶解的结晶紫的吸光度。






图2 。念珠菌的生物膜形成。A)硅橡胶表面的图像B)在8%柠檬草油涂层的硅橡胶表面上形成生物膜C)在0%柠檬草油涂层的硅橡胶表面上形成生物膜(仅羟丙基甲纤维素软膏)D)在没有涂层的硅橡胶上形成生物膜(阳性对照) )。


数据分析


设置没有任何涂层和微生物的硅橡胶表面的吸光度值,但将其与生物膜生长和结晶紫染色的步骤相同,作为阴性对照。从具有生物膜的表面的吸光度值中减去该值。
评估没有涂层的硅橡胶表面上生物膜的形成,作为阳性对照,并将其设置为100%。将涂有羟丙甲纤维素软膏且无任何精油的硅橡胶表面设置为0%精油涂覆的表面。
使用以下等式,测量处理物生物膜形成的吸光度值,并计算处理物生物膜形成相对于阳性对照的减少量(%):


降低百分比= [((吸光度阳性对照(560 nm)–吸光度处理(560 nm))/吸光度阳性对照(560 nm)] × 100%


使用MINITAB 18软件进行统计分析。
应用安德森-达林测试以确定是否生物膜形成和生物膜抑制数据瓦特ERE正态分布的。此后,应用Levene检验来评估方差的均匀性。
如果发现结果呈正态分布,则应用两因素实验设计并通过Tukey测试进行成对比较。如果未发现结果呈正态分布,请使用非参数检验(Kruskal-Wallis和Mann-Whitney U检验)评估结果。


笔记


至少需要一式三份地通过修饰的结晶紫结合测定法确定精油包被的硅橡胶表面上念珠菌的生物膜形成,包括对照。
当吸光度太高时,将溶解的结晶紫稀释,然后将吸光度值乘以稀释倍数。


菜谱


脑心脏输液肉汤(1升)
将37 g BHI粉末溶于1 L蒸馏水中,并使用高压釜在121°C的温度下灭菌15分钟。


脑心浸液琼脂(1升)
在1 L蒸馏水中溶解37 g BHI粉末和30 g Agar-Agar粉末,然后在121°C下用高压釜灭菌15分钟后,将培养基放入无菌的陪替氏培养皿中。


1%结晶紫溶液(w / v)
将5 g结晶紫溶解在500 ml蒸馏水中,并将结晶紫溶液(1%,w / v)在摇动培养箱中于室温以150 rpm孵育2 h 。


10 mM磷酸钾缓冲液(pH 7.0)(1 L)
将4.35 g K 2 HPO 4和3.4 g KH 2 PO 4溶解在100 ml蒸馏水中,得到储备的0.5 M磷酸钾缓冲液。
将0.5 M磷酸钾缓冲液稀释至10 mM磷酸钾缓冲液。
使用pH计将缓冲液的pH值调节到7.0,并用高压灭菌器在121°C下消毒15分钟。


致谢


这项研究得到了土耳其安卡拉哈塞特佩大学科研项目协调部(授权号:FDK-2016-10821)的资助。该协议源自Sahal等人。(2020)。


利益争夺


该作者宣称没有竞争的经济利益。


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引用:Sahal, G., Woerdenbag, H. J., Hinrichs, W., Visser, A., van der Mei, H. C. and Bilkay, I. S. (2021). Candida Biofilm Formation Assay on Essential Oil Coated Silicone Rubber. Bio-protocol 11(5): e3941. DOI: 10.21769/BioProtoc.3941.
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