Abstract
Objective
Root canal irrigation is an important adjunct to control microbial infection. This study aimed primarily to develop a transparent root canal model to study in situ Enterococcus faecalis biofilm removal rate and remaining attached biofilm using passive or active irrigation solution for 90 s. The change in available chlorine and pH of the outflow irrigant were assessed.
Methods
A total of forty root canal models ( n = 10 per group) were manufactured using 3D printing. Each model consisted of two longitudinal halves of an 18 mm length simulated root canal with size 30 and taper 0.06. E. faecalis biofilms were grown on the apical 3 mm of the models for 10 days in Brain Heart Infusion broth. Biofilms were stained using crystal violet for visualization. The model halves were reassembled, attached to an apparatus and observed under a fluorescence microscope. Following 60 s of 9 mL of 2.5% NaOCl irrigation using syringe and needle, the irrigant was either left stagnant in the canal or activated using gutta-percha, sonic and ultrasonic methods for 30 s. Images were then captured every second using an external camera. The residual biofilm percentages were measured using image analysis software. The data were analyzed using Kruskal–Wallis test and generalized linear mixed model.
Results
The highest level of biofilm removal was with ultrasonic agitation (90.13%) followed by sonic (88.72%), gutta-percha (80.59%), and passive irrigation group (control) (43.67%) respectively. All agitation groups reduced the available chlorine and pH of NaOCl more than that in the passive irrigation group.
Significance
The 3D printing method provided a novel model to create a root canal simulation for studying and understanding a real-time biofilm removal under microscopy. Ultrasonic agitation of NaOCl left the least amount of residual biofilm in comparison to sonic and gutta-percha agitation methods.
1
Introduction
Root canal treatment describes the dental procedure used to either prevent apical periodontitis by the treatment of diseased or infected soft tissue contained in the root canal system, or the procedure used to resolve established apical periodontitis , which is caused mainly by bacteria . Bacteria adhere to the root canal surfaces and rapidly form biofilms . A biofilm is defined as a community of microorganisms of one or more species embedded in an extracellular polysaccharide matrix that is attached to a solid substrate . Thus, the essential aim of the root canal treatment involves the microbial control of the root canal system through instrumentation and irrigation. Instrumentation aims to give the canal system a shape that permits the delivery of locally used medications ( e.g. , irrigant), as well as a root canal filling, which helps to entrap the remaining microbiota . Irrigation also aims to lubricate the instruments, and, remove pathogenic microorganisms (microbiota) in the root canal system through the flushing action . However, as the lubricated instrument is rotated along its long axis to sculpt the inner canal surface which it engages with, the most apical part of the canal remains untouched . Thus, the use of a final irrigation regimen, after the completion of a chemo-mechanical canal preparation, with high volumes of various chemically active solutions may contribute to removing residual biofilm in the non-instrumented part of the root canal system .
The debridement action of an irrigant within the root canal system may remain elusive when using a needle and syringe alone . Two phenomena are inherent to irrigant penetration and debridement action in the confined space of a closed root canal system. First, the stagnation of the irrigant flow beyond the irrigation needle tip . Second, the gas bubbles or vapor locks effect ahead of the advancing front of the irrigant . These phenomena may limit the delivery of irrigant to the canal terminus . For the above mentioned reasons, attempts to improve the efficacy of irrigant penetration and irrigant mixing within the root canal system are therefore important since they may improve the removal of residual biofilms. Irrigant agitation may be applied to aid the dispersal of the irrigant into the root canal system, especially into the periapical terminus of the canal . Agitation techniques for root canal irrigants include either manual agitation or automated agitation .
Manual agitation of the irrigant could be achieved by using a file or a tapered gutta-percha cone , which is achieved by moving the master file or gutta-percha cone up and down in short strokes within an instrumented canal . Automated devices for agitation of the irrigant in the root canal system include ultrasonic and sonic devices .
During ultrasonic agitation, a file oscillates at frequencies of 25–30 kHz in a pattern of motion consisting of nodes and anti-nodes along its length . During sonic agitation of the irrigant, the file oscillates at frequencies of 1–6 kHz , and it produces lower shear stresses compared to ultrasonic agitation . The EndoActivator system is a sonic device with polymer tips with a cordless electrically driven hand-piece .
The issue of the efficacy of irrigation protocol to remove bacterial biofilm has received considerable critical attention. It has been investigated by the immersing of samples in a static irrigant that neglect irrigant flow within the confinement of a root canal system . Other studies used Computational Fluid Dynamics models to measure the physical parameters associated with irrigant flow within the root canal system, that lack the ability to estimate the chemical action of irrigant as they provide a virtual view of the root canal irrigation .
Although the use of extracted teeth might be clinically relevant, it may not be the optimum method as the root canal components (dentin, cementum) are concealed body compartments , making them unavailable for direct visualization. In addition, the use of extracted teeth of a different size introduces many variables to the studies .
Attempts to mimic the root canal anatomy using gypsum converted to hydroxyapatite have shown promising anatomical features, but such opaque materials did not allow direct visualization. The use of 3D printing models to study root canal disinfection has been explored in a preliminary study , but the tested steriolithography material, Visijet ® EX200 Plastic did not allow bacterial colonization and was not transparent. It seems justifiable to develop an in vitro model that provides transparency and generation of multiple samples with the same anatomical features to investigate the real-time interaction between the activated irrigant and biofilm removal during the irrigation process.
This study aimed primarily to develop and utilize transparent test models to facilitate an investigation into the influence of NaOCl agitation on the removal rate of Enterococcus faecalis biofilm subjected to sodium hypochlorite irrigation. A further aim was to compare the residual biofilm and removal rate of biofilm when subjected to passive (stagnant) and active irrigation (2.5% NaOCl). Finally, the outcomes of chemical interaction between a NaOCl irrigant and bacterial biofilm ( in situ ) represented by the available chlorine and pH of outflow irrigant, as outcome measures were assessed.
2
Materials and methods
2.1
Construction of transparent root canal models and distribution to experimental groups
A solid computer representation of the root canal model was created using AutoCAD ® software (Autodesk, Inc., San Rafael, CA, USA). The design of the model consisted of two equal rectangular molds (18 mm × 6 mm × 1 mm) ( Fig. 1 ).
Each mold contained four holes on either side, as well as a longitudinal half canal. When the two molds were reassembled, a straight simple canal of 18 mm length, apical size 30, and a 0.06 taper was created.
The AutoCAD format of the model was converted into stereo-lithography format (STL format). Forty root canal models were manufactured using PreForm Software 1.9.1 of Formlabs 3D printer (Formlabs Inc., Somerville, MA, USA). The material used to create the model was a clear liquid photopolymer material (AZoNetwork Ltd., Cheshire, UK). It is composed of a mixture of methacrylates and a photo-initiator. The process of fabrication started by conversion of the digital geometric data of the model into a series of layers that were physically constructed layer-by-layer of 25-μm thickness. Each layer was fabricated by exposing the liquid photopolymer material to a laser light source from the printer causing the liquid to cure into a transparent solid state.
The models ( n = 40) divided to four groups ( n = 10 per group) ( Table 1 ). In the passive irrigation group, the irrigant was delivered using a 10 mL syringe (Plastipak, Franklin Lakes, New Jersey, USA) with a 27-gauge side-cut open-ended needle (Monoject, Sherwood Medical, St. Louis, MO, USA). In the gutta-percha (GP) irrigation group, the irrigant was delivered as in the previous group and agitated using a cone GP (SybronEndo, Buffalo, New York, USA). In the sonic irrigation group, the irrigant was delivered as in the first group but agitated using the EndoActivator ® device (Dentsply Tulsa Dental Specialities, Tulsa, OK, USA). In the ultrasonic irrigation group, the irrigant was delivered as in the first group but agitated using a Satelec ® P5 ultra-sonic device (Satelec, Acteon, Equipment, Merignac, France).
| Group | Type of irrigation |
|---|---|
| Passive irrigation ( n = 10) | Syringe and needle + passive irrigant stagnation |
| GP irrigation ( n = 10) | Syringe and needle + GP irrigation |
| Sonic irrigation ( n = 10) | Syringe and needle + sonic irrigation |
| Ultrasonic irrigation ( n = 10) | Syringe and needle + ultrasonic irrigation |
2.2
Generation of single species biofilm ( E. faecalis ) on the surface of the canal models
2.2.1
Preparation of microbial strain and determination of the standard inoculum (CFU/mL)
Biofilms were grown from a single bacterial strain ( E. faecalis; ATCC 19433). The strain was supplied in the form of frozen stock in a brain-heart infusion broth (BHI) (Sigma–Aldrich, USA) and 30% glycerol stored at −70 °C. The strain was thawed to a temperature of 37 °C for 10 min and swirled for 30 s . After thawing, 100 μL of the strain were taken and plated onto a BHI agar plate (Sigma–Aldrich, St. Louis, Montana, USA) with 5% defibrinated horse blood (E&O Laboratories, Scotland, UK) and incubated at 37 °C in the 5% CO 2 incubator (LEEC, Nottingham, UK) for 24 h. Bacterial morphology and catalase activity were confirmed prior to the generation of the biofilms. For this, two colonies of the strain were separately removed using a sterile inoculating loop (VWR, Leicester, UK), and catalase testing using 3% H 2 O 2 (Sigma–Aldrich Ltd., Dorset, UK) and Gram-staining (BD Ltd., Oxford, UK) were performed.
A standard inoculum was used. For this, six colonies were removed from the agar plate, placed into 20 mL of BHI broth, and incubated at 37 °C in a 5% CO 2 incubator for 24 h. BHI containing E. faecalis was adjusted to 0.5 absorbance at a wavelength of 600 nm using a spectrophotometer (NanoDrop™ Spectrophotometer ND-100, Wilmington, USA) . Inoculum concentration was confirmed using a total of six ten-fold serial dilutions to determine the colony forming units per milliliter (CFUs/mL) corresponding to 1.1 × 10 8 CFU/mL.
2.2.2
Sterilization of the canal models
The model halves were packed individually in packaging bags (Sterrad 100S, ASP ® , Irvine, CA, USA) and then sterilized using gas plasma with hydrogen peroxide vapor (Sterrad 100S, ASP ® , Irvine, CA, USA) for 50 min .
2.2.3
Generation and staining of E. faecalis biofilm on the canal surface
One mL of standard E. faecalis inoculum was delivered into a sterilized 7 mL plastic bijou bottle (Sarstedt Ltd., Nümbrecht, Germany) that contained a single sterilized half model such that the 3 mm apical portion was immersed. This was achieved using a sterile syringe (BD Plastipak™, Franklin Lakes, NJ, USA) and a 21-gauge needle (BD Microlance™, Franklin Lakes, NJ, USA) to insert the inoculum. The samples were then incubated at 37 °C in the 5% CO 2 incubator for 10 days. Every 2 days, half of the inoculum that surrounded the sample was discarded and replaced with fresh BHI broth .
After ten days incubation, all samples with biofilms were removed from the plastic bottle and prepared for staining with a crystal violet dye (CV) . The model halves containing the biofilms were placed onto a slide facing up and rinsed with 1 mL sterile distilled water (Roebuck, London, UK) for 1 min using a sterile 10 mL syringe (Plastipak, Franklin Lakes, New Jersey, USA) to remove loosely attached cells. Using a micropipette (Alpha Laboratories Ltd., Eastleigh, Winchester, UK), 1 μL of CV stain (Merck, Darmstadt, Germany) was applied in the apical 3 mm of the model and left for 1 min to allow staining. The stained canals were subsequently washed with 3 mL of sterile distilled water for 1 min . Subsequently, the models were re-assembled for the irrigation experiments as described below.
2.3
Re-apposition of the model halves
Before reassembling the two model halves, a polyester seal film of 0.05 mm thickness (UnisealTM, Buckingham, UK) was positioned on the half coated with biofilm. Any part of the film that overhung the canal boundary was removed using a surgical blade (Swann-Morton, Sheffield, UK) without disturbing the biofilm. The two halves of the model were then held in position using four brass bolts (size 16 BA) and nuts (Clerkenwell Screws, London, UK).
2.4
Irrigation experiments
The apical end of each canal was blocked using a sticky wax (Associated Dental Product Ltd., Swindon, UK). Each model was fixed to a plastic microscopic slide (75 × 25 × 1.2 mm) (Fisher Scientific Ltd., Rochester, NY, USA) using a custom-fabricated clamp. The model half with the biofilm faced the slide. The microscopic slide was placed on a stage of an inverted fluorescence microscope (Leica, UK). The test irrigant used in experiments was NaOCl (Teepol ® Bleach, UK).
Concentration of available NaOCl was verified before experiments using iodometric titration (British Pharmacopoeia 1973) and adjusted to 2.5%. A total of 9 mL of irrigant (NaOCl) were delivered using a 10 mL syringe (Plastipak, Franklin Lakes, New Jersey, USA) with a 27-gauge side-cut open-ended needle (Monoject, Sherwood Medical, St. Louis, MO, USA). The needle was inserted 3 mm coronal to the canal terminus. The port opening of the needle always faced the model half containing the biofilm. The syringe was attached to a programmable precision syringe pump (NE-1010) to deliver the irrigant in 60 s at a flow rate of 0.15 mL s −1 , followed by 30 s of irrigant that was either kept stagnant (passive) in the canal or activated using GP, sonic and ultrasonic methods.
For the GP agitation group, a gutta-percha cone with an apical ISO size 30 and 0.02 taper was placed 2 mm coronal to the canal terminus which was used to agitate the irrigant in the root canal system with a push–pull amplitude of approximately 3–5 mm at a frequency of 50 strokes per 30 s. A new GP cone was used with each canal model.
For the sonic agitation group, the agitation was carried out using an EndoActivator ® device by placing the polymer tip of an EndoActivator ® device with size 25 and 0.04 taper at 2 mm from the canal terminus, and then the agitation was continued for 30 s with high power-setting (Ruddle ). Once again, a new tip was used with each canal model.
For the ultrasonic agitation group, the agitation was carried out by placing a stainless steel instrument size and taper 20/02 (IrriSafe; Satelec Acteon, Merignac, France) of Satelec ® P5 Newtron piezon unit at 2 mm from the canal terminus, then the agitation was continued for 30 s. The file was energized at power setting 7 as recommended by the manufacturer. A new tip was used with each canal model.
Outflow irrigant was collected in a 15 mL plastic tube (TPP, Schaffhausen, Switzerland) using a vacuum pump (Neuberger, London, UK) ( Fig. 2 ). The amount of available chlorine (%) and pH of the outflow NaOCl were measured using iodometric titration (British Pharmacopoeia 1973) and a pH calibration meter (HANNA pH 211, Hanna Instrument, UK) respectively.
2.5
Recording of biofilm removal by the irrigation procedure
Removal of biofilm was recorded using a high-resolution CCD camera (QICAM, Canada). The camera was connected to a 2.5× lens of a fluorescence microscope (Leica, UK). An N2.1 longpass filter was used during the time-lapse recording of interactions between the irrigant and the biofilm.
2.6
Image analysis
One video per irrigation procedure was obtained and images were captured at each second of footage (90 images). The canal surface coverage of biofilm present after every second of irrigation (0.15 mL) was visualized and quantified using Image-pro Plus 4.5 software (MediaCybernetics ® , Silver Springs, New York, USA) ( Fig. 3 ).
2.7
Data analyses
The residual biofilm (%) at each second of 90 s irrigation with passive and active NaOCl irrigation was analyzed using line plots. An assumption concerning a normal distribution of data for the residual biofilm was checked using a visual inspection of the box and whisker plots. The data representing the percentages of residual biofilm covering the canal surface area were not normally distributed and therefore the non-parametric Kruskal–Wallis test, followed by Bonferroni post-hoc comparisons were performed to compare their distributions in the four experimental groups. The effects of irrigant agitation duration on the percentage of residual biofilm covering the canal surface area were analyzed by the type of irrigation (passive or GP, sonic, and ultrasonic active irrigation) using a generalized linear mixed model. The differences in median of chlorine and pH values of the outflow NaOCl of the four groups before and after irrigation were compared using the Kruskal–Wallis test. A significance level of 0.05 was used throughout. The data were analyzed by SPSS (BM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, New York, IBM Corp.).
2
Materials and methods
2.1
Construction of transparent root canal models and distribution to experimental groups
A solid computer representation of the root canal model was created using AutoCAD ® software (Autodesk, Inc., San Rafael, CA, USA). The design of the model consisted of two equal rectangular molds (18 mm × 6 mm × 1 mm) ( Fig. 1 ).
Each mold contained four holes on either side, as well as a longitudinal half canal. When the two molds were reassembled, a straight simple canal of 18 mm length, apical size 30, and a 0.06 taper was created.
The AutoCAD format of the model was converted into stereo-lithography format (STL format). Forty root canal models were manufactured using PreForm Software 1.9.1 of Formlabs 3D printer (Formlabs Inc., Somerville, MA, USA). The material used to create the model was a clear liquid photopolymer material (AZoNetwork Ltd., Cheshire, UK). It is composed of a mixture of methacrylates and a photo-initiator. The process of fabrication started by conversion of the digital geometric data of the model into a series of layers that were physically constructed layer-by-layer of 25-μm thickness. Each layer was fabricated by exposing the liquid photopolymer material to a laser light source from the printer causing the liquid to cure into a transparent solid state.
The models ( n = 40) divided to four groups ( n = 10 per group) ( Table 1 ). In the passive irrigation group, the irrigant was delivered using a 10 mL syringe (Plastipak, Franklin Lakes, New Jersey, USA) with a 27-gauge side-cut open-ended needle (Monoject, Sherwood Medical, St. Louis, MO, USA). In the gutta-percha (GP) irrigation group, the irrigant was delivered as in the previous group and agitated using a cone GP (SybronEndo, Buffalo, New York, USA). In the sonic irrigation group, the irrigant was delivered as in the first group but agitated using the EndoActivator ® device (Dentsply Tulsa Dental Specialities, Tulsa, OK, USA). In the ultrasonic irrigation group, the irrigant was delivered as in the first group but agitated using a Satelec ® P5 ultra-sonic device (Satelec, Acteon, Equipment, Merignac, France).
| Group | Type of irrigation |
|---|---|
| Passive irrigation ( n = 10) | Syringe and needle + passive irrigant stagnation |
| GP irrigation ( n = 10) | Syringe and needle + GP irrigation |
| Sonic irrigation ( n = 10) | Syringe and needle + sonic irrigation |
| Ultrasonic irrigation ( n = 10) | Syringe and needle + ultrasonic irrigation |
2.2
Generation of single species biofilm ( E. faecalis ) on the surface of the canal models
2.2.1
Preparation of microbial strain and determination of the standard inoculum (CFU/mL)
Biofilms were grown from a single bacterial strain ( E. faecalis; ATCC 19433). The strain was supplied in the form of frozen stock in a brain-heart infusion broth (BHI) (Sigma–Aldrich, USA) and 30% glycerol stored at −70 °C. The strain was thawed to a temperature of 37 °C for 10 min and swirled for 30 s . After thawing, 100 μL of the strain were taken and plated onto a BHI agar plate (Sigma–Aldrich, St. Louis, Montana, USA) with 5% defibrinated horse blood (E&O Laboratories, Scotland, UK) and incubated at 37 °C in the 5% CO 2 incubator (LEEC, Nottingham, UK) for 24 h. Bacterial morphology and catalase activity were confirmed prior to the generation of the biofilms. For this, two colonies of the strain were separately removed using a sterile inoculating loop (VWR, Leicester, UK), and catalase testing using 3% H 2 O 2 (Sigma–Aldrich Ltd., Dorset, UK) and Gram-staining (BD Ltd., Oxford, UK) were performed.
A standard inoculum was used. For this, six colonies were removed from the agar plate, placed into 20 mL of BHI broth, and incubated at 37 °C in a 5% CO 2 incubator for 24 h. BHI containing E. faecalis was adjusted to 0.5 absorbance at a wavelength of 600 nm using a spectrophotometer (NanoDrop™ Spectrophotometer ND-100, Wilmington, USA) . Inoculum concentration was confirmed using a total of six ten-fold serial dilutions to determine the colony forming units per milliliter (CFUs/mL) corresponding to 1.1 × 10 8 CFU/mL.
2.2.2
Sterilization of the canal models
The model halves were packed individually in packaging bags (Sterrad 100S, ASP ® , Irvine, CA, USA) and then sterilized using gas plasma with hydrogen peroxide vapor (Sterrad 100S, ASP ® , Irvine, CA, USA) for 50 min .
2.2.3
Generation and staining of E. faecalis biofilm on the canal surface
One mL of standard E. faecalis inoculum was delivered into a sterilized 7 mL plastic bijou bottle (Sarstedt Ltd., Nümbrecht, Germany) that contained a single sterilized half model such that the 3 mm apical portion was immersed. This was achieved using a sterile syringe (BD Plastipak™, Franklin Lakes, NJ, USA) and a 21-gauge needle (BD Microlance™, Franklin Lakes, NJ, USA) to insert the inoculum. The samples were then incubated at 37 °C in the 5% CO 2 incubator for 10 days. Every 2 days, half of the inoculum that surrounded the sample was discarded and replaced with fresh BHI broth .
After ten days incubation, all samples with biofilms were removed from the plastic bottle and prepared for staining with a crystal violet dye (CV) . The model halves containing the biofilms were placed onto a slide facing up and rinsed with 1 mL sterile distilled water (Roebuck, London, UK) for 1 min using a sterile 10 mL syringe (Plastipak, Franklin Lakes, New Jersey, USA) to remove loosely attached cells. Using a micropipette (Alpha Laboratories Ltd., Eastleigh, Winchester, UK), 1 μL of CV stain (Merck, Darmstadt, Germany) was applied in the apical 3 mm of the model and left for 1 min to allow staining. The stained canals were subsequently washed with 3 mL of sterile distilled water for 1 min . Subsequently, the models were re-assembled for the irrigation experiments as described below.
2.3
Re-apposition of the model halves
Before reassembling the two model halves, a polyester seal film of 0.05 mm thickness (UnisealTM, Buckingham, UK) was positioned on the half coated with biofilm. Any part of the film that overhung the canal boundary was removed using a surgical blade (Swann-Morton, Sheffield, UK) without disturbing the biofilm. The two halves of the model were then held in position using four brass bolts (size 16 BA) and nuts (Clerkenwell Screws, London, UK).
2.4
Irrigation experiments
The apical end of each canal was blocked using a sticky wax (Associated Dental Product Ltd., Swindon, UK). Each model was fixed to a plastic microscopic slide (75 × 25 × 1.2 mm) (Fisher Scientific Ltd., Rochester, NY, USA) using a custom-fabricated clamp. The model half with the biofilm faced the slide. The microscopic slide was placed on a stage of an inverted fluorescence microscope (Leica, UK). The test irrigant used in experiments was NaOCl (Teepol ® Bleach, UK).
Concentration of available NaOCl was verified before experiments using iodometric titration (British Pharmacopoeia 1973) and adjusted to 2.5%. A total of 9 mL of irrigant (NaOCl) were delivered using a 10 mL syringe (Plastipak, Franklin Lakes, New Jersey, USA) with a 27-gauge side-cut open-ended needle (Monoject, Sherwood Medical, St. Louis, MO, USA). The needle was inserted 3 mm coronal to the canal terminus. The port opening of the needle always faced the model half containing the biofilm. The syringe was attached to a programmable precision syringe pump (NE-1010) to deliver the irrigant in 60 s at a flow rate of 0.15 mL s −1 , followed by 30 s of irrigant that was either kept stagnant (passive) in the canal or activated using GP, sonic and ultrasonic methods.
For the GP agitation group, a gutta-percha cone with an apical ISO size 30 and 0.02 taper was placed 2 mm coronal to the canal terminus which was used to agitate the irrigant in the root canal system with a push–pull amplitude of approximately 3–5 mm at a frequency of 50 strokes per 30 s. A new GP cone was used with each canal model.
For the sonic agitation group, the agitation was carried out using an EndoActivator ® device by placing the polymer tip of an EndoActivator ® device with size 25 and 0.04 taper at 2 mm from the canal terminus, and then the agitation was continued for 30 s with high power-setting (Ruddle ). Once again, a new tip was used with each canal model.
For the ultrasonic agitation group, the agitation was carried out by placing a stainless steel instrument size and taper 20/02 (IrriSafe; Satelec Acteon, Merignac, France) of Satelec ® P5 Newtron piezon unit at 2 mm from the canal terminus, then the agitation was continued for 30 s. The file was energized at power setting 7 as recommended by the manufacturer. A new tip was used with each canal model.
Outflow irrigant was collected in a 15 mL plastic tube (TPP, Schaffhausen, Switzerland) using a vacuum pump (Neuberger, London, UK) ( Fig. 2 ). The amount of available chlorine (%) and pH of the outflow NaOCl were measured using iodometric titration (British Pharmacopoeia 1973) and a pH calibration meter (HANNA pH 211, Hanna Instrument, UK) respectively.
