A portable extensional rheometer for measuring the viscoelasticity of pitcher plant and other sticky liquids in the field

A portable extensional rheometer for measuring the viscoelasticity of pitcher plant and other sticky liquids in the field Collett et al. PLANT METHODS Collett et al. Plant Methods (2015) 11:16 DOI 10.1186/s13007-015-0059-5 METHODOLOGY Open Access A portable extensional rheometer for measuring the viscoelasticity of pitcher plant and other sticky liquids in the field Catherine Collett 1 , Alia Ardron 1 , Ulrike Bauer 2,3 , Gary Chapman 1 , Elodie Chaudan 1,4 , Bart Hallmark 1 , Lee Pratt 1 , Maria Dolores Torres-Perez 1,5 and D Ian Wilson 1* Abstract Background: Biological fluids often have interesting and unusual physical properties to adapt them for their specific purpose. Laboratory-based rheometers can be used to characterise the viscoelastic properties of such fluids. This, however, can be challenging as samples often do not retain their natural properties in storage while conventional rheometers are fragile and expensive devices ill-suited for field measurements. We present a portable, low-cost extensional rheometer designed specifically to enable in situ studies of biological fluids in the field. The design of the device (named Seymour) is based on a conventional capillary break-up extensional rheometer (the Cambridge Trimaster). It works by rapidly stretching a small fluid sample between two metal pistons. A battery-operated solenoid switch triggers the pistons to move apart rapidly and a compact, robust and inexpensive, USB 3 high speed camera is used to record the thinning and break-up of the fluid filament that forms between the pistons. The complete setup runs independently of mains electricity supply and weighs approximately 1 kg. Post-processing and analysis of the recorded images to extract rheological parameters is performed using open source software. Results: The device was tested both in the laboratory and in the field, in Brunei Darussalam, using calibration fluids (silicone oil and carboxymethyl cellulose solutions) as well as Nepenthes pitcher plant trapping fluids as an example of a viscoelastic biological fluid. The fluid relaxation times ranged from 1 ms to over 1 s. The device gave comparable performance to the Cambridge Trimaster. Differences in fluid viscoelasticity between three species were quantified, as well as the change in viscoelasticity with storage time. This, together with marked differences between N. rafflesiana fluids taken from greenhouse and wild plants, confirms the need for a portable device. Conclusions: Proof of concept of the portable rheometer was demonstrated. Quantitative measurements of pitcher plant fluid viscoelasticity were made in the natural habitat for the first time. The device opens up opportunities for studying a wide range of plant fluids and secretions, under varying experimental conditions, or with changing temperatures and weather conditions. Keywords: Biological fluids, Filament, Giesekus, Nepenthes, Pitcher plants, Polymer solution, Polysaccharide, Rheometry * Correspondence: [email protected] 1 Department of Chemical Engineering and Biotechnology, New Museums Site, Pembroke Street, Cambridge CB2 3RA, UK Full list of author information is available at the end of the article PLANT METHODS © 2015 Collett et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Collett et al. Plant Methods (2015) 11:16 DOI 10.1186/s13007-015-0059-5 Background Viscoelastic behaviour of biological fluids Water has long been recognised as the essence of life, and many ubiquitous biological fluids such as cytoplasm, blood and plant sap are based on water. In contrast to pure water, aqueous (and other) biological fluids often exhibit non-Newtonian behaviour such as shear-thinning (e.g. blood [1], bronchial mucus [2], gastropod foot mucus [3] and the adhesive fluids of insects [4]). Soluble long chain polymers give rise to viscoelastic behaviour and the ability to form filaments of liquid that can stretch [5]. This impacts a broad range of biological processes from the locomotion of sperm through cervical mucus [6] to the spinning of spider silk [7] and the trapping of insects by carnivorous plants (genera Drosera [8], Drosophyllum,Pinguicula and Nepenthes [9]). Limitations of current rheometry methods Accurate measurement of viscoelastic fluid properties, using extensional rheometry, is essential for understanding their contribution to the biological function. As part of living organisms, biological fluids often undergo marked changes over time [10,11], and fluid properties need to be monitored at short intervals in the natural environment in order to investigate these dynamic processes and their effects. Rigorous quantitative measurements are currently not possible in the field (laboratory devices are expensive, fragile and not readily transported), while the viscoelastic properties of many natural liquids change after sampling. Resins and latex are examples which change properties rapidly when exposed to air. Furthermore, the fluid properties depend on environmental factors such as temperature and air humidity, while the size and immobility of traditional extensional rheometers prohibits their use in climate control chambers. There is therefore a need for a portable device to study viscoelastic biological fluids in situ or under controlled environmental conditions. This paper reports the development of such a device, which arose from the desire to study pitcher plant fluids in situ in Borneo (it was consequently named Seymour after the owner of a carnivorous plant in the movie ‘Little Shop of Horrors’). The device can be used for routine testing as well as field work. It offers the following advantages: (a) It is lightweight, robust, easy to assemble and has few moving parts; (b) It is constructed mainly from standard parts, which can be replaced readily, and is therefore relatively inexpensive; (c) It employs small sample volumes (<10 μL), which fits the increasing demand for the miniaturisation of rheometric techniques owing to the limited availability or high cost of samples [12]; (d) It is easy to operate. Data can be analysed directly or remotely; (e) It is suitable for testing over a broad range of temperatures and humidity levels as it fits readily into a controlled environment chamber: many studies of extensional rheology to date have been limited to standard laboratory conditions [13]. Pitcher plant fluids We used pitcher plant fluids as an example of a viscoelastic biological fluid in order to test the performance of the Seymour device in the laboratory and in the field. Nepenthes pitcher plant fluids are sticky aqueous solutions of polysaccharides [9] held in cupshaped leaves to trap insects. Prey struggling at the fluid surface quickly cover themselves in sticky threads, much like a piece of bread is covered by molten cheese in a fondue. The plant subsequently digests the drowned insects to release and absorb mineral nutrients. Bauer et al. [14] compared different pitcher plant species in the field using a crude measure of extensional viscosity, namely the length of filament that could be formed by stretching the fluid between two fingers. In addition to marked differences in apparent fluid viscoelasticity between species they found strong variation between individual plants. Observations on greenhouse plants of N. rafflesiana further suggest that greenhouse cultivation affects fluid viscoelasticity negatively (Bauer, unpublished). It is not clear whether this is a result of suboptimal growth conditions leading to reduced polysaccharide production by the plant, or due to dilution of the pitcher fluid when the plants are watered. Erni et al. [8] studied the extensional behaviour of Drosera mucilage using a microscope-based CaBER™ device and reported phenomena such as beads-onstring formation associated with viscoelastic fluids, with a relaxation time of circa 0.33 s. Gaume and Forterre [9] measured the filament thinning of N. rafflesiana fluid with a simple rod arrangement and reported viscoelastic behaviour with a relaxation time of circa 1 s. This relaxation time was longer than the period in which insect prey were observed to flex limbs and was therefore interpreted as an adaptation for prey capture. Gaume and Forterre collected fluid samples in the field [9] but it is not clear if the viscoelastic testing was performed on location. Their method also required a pool of liquid to withdraw the rod from, which is not feasible for smaller sample volumes such as the mucilage droplets of Drosera,Drosophyllum and Pinguicula. Collett et al. Plant Methods (2015) 11:16 Page 2 of 16 Extensional rheometry Extensional rheology is the study of the deformation of material under conditions of pure strain; in this paper the exact form of the strain is the Hencky strain. The more commonly studied case is that of deformation under pure shear, where a sample is placed between two surfaces which move past one another, keeping the separating distance constant (or where the fluid is allowed to flow along a pipe so that the fluid shears against itself). Extensional rheometry requires the material to be stretched, and generating a reproducible stretching requires a precise mechanical action. Unlike Newtonian liquids, the extensional behaviour of viscoelastic fluids cannot be estimated reliably from measurements of shear rheology so direct measurements are necessary. There are several methods for measuring extensional rheology [5]. Filament stretching is now a routine method in the laboratory, using devices such as the FiSER™(Cambridge Polymer Group, Boston), CaBER™ [15] (Cambridge Polymer Group, Boston and Haake), and the Cambridge Trimaster [16] to measure the necking of an extensionally-strained fluid filament as a function of time. The evolution of the filament diameter can be used to identify the nature of the fluid (e.g. Newtonian, viscoelastic etc., see next paragraph) and, given the surface tension of the fluid, to calculate the extensional viscosity. Viscoelastic fluids are characterised by viscous and elastic contributions: an important parameter in the latter is the relaxation time, λ, which is simply speaking the time needed for the polymer molecules to adjust to a change in strain. A review of filament stretching rheometers can be found in Galindo-Rosales et al. [17]. A sample of liquid is placed between two platens (i.e. the ends of two rods), and the platens moved apart either (i)atcontrolled separating speed, imparting a known strain rate, or (ii) rapidly, causing the formation of a filament which thins under capillary action. The latter mode, shown in Figure 1(a), known as CaBER™(capillary break-up extensional rheometry [18]) mode, is used here. A high speed camera and image analysis software monitors the filament diameter at its mid-point, D,overtime,t.The filament will break at the mid-point under pure extension. Gravity has negligible effect with the devices employed here, as the dimensions give Bond numbers « 1 [9]; this is discussed after Equation (7). The evolution of Dwith respect to its initial value, D 0 , has been determined for various constitutive fluid models, and those considered here are: (a) Newtonian liquid, with constant shear viscosity, η 0 , and surface tension, α[19]. A correction factor, X, has been included to account for the non-cylindrical nature of the fluid filament: X=1 for ideal, cylindrical filaments; X= 0.7127 for non-inertial, smooth, filaments. More information on this correction, and on the systematic differences in Newtonian viscosity calculated by extensional and shear measurements, can be found in McKinley and Tripathi [15] and Liang and Mackley [20]. D D0 ¼1‐ 2X−1ðÞαt 3η0D0 ð1Þ (b) The Upper Convected Maxwell (UCM) model, representing the simplest viscoelastic fluid model [19] for negligible viscosity with relaxation time, λ UCM ; D D0 ¼exp −t 3λUCM  ð2Þ (c) The Giesekus model for viscoelastic solutions [21], which includes a relaxation time, λ G , and a polymer 1.2 mm (a) (b) (c) Figure 1 Schematic of CaBER™operation. (a) standard mode, platens move equal distance apart. Initial gap g 0 , final gap g f .(b) Seymour operation, only upper platen moves. (c) Example of N. maxima filament shortly before breakup, g 0 = 0.4 mm. Time set to zero when platens stop moving, (ii); filament width measured (iii) until break up at t F ,(iv). Collett et al. Plant Methods (2015) 11:16 Page 3 of 16 interaction term, the mobility parameter, a, gives the following implicit relationship for D/D 0 [22]; 4a−3ðÞln D=D0þ2αλGa=η0D0 1þ2αλGa=η0D0  − 2D0η0 αλG D=D0−1ðÞ¼ t λG ð3Þ Numerical fitting techniques are needed to extract the Giesekus model parameters from filament thinning data sets. As the filament thins it will break due to capillary instability. The time where this occurs will depend on the nature of the filament but an estimate of the filament break-up time, t F , can be obtained by setting D/D 0 =0. For a Newtonian fluid, Equation (1) gives tF≈3η0D0 α2X−1ðÞ ð4Þ The Seymour concept In the Trimaster (and other devices), the platens move apart so that the filament midpoint remains at the same location, greatly reducing the computational effort in analysing images but requiring delicate mechanical action (Figure 1(a)). In contrast, the Seymour only moves one platen using a standard solenoid switch (Figure 1 (b)). This improves the robustness of the device (fewer moving parts) and reduces the cost demonstrably. The major cost component is the digital camera: the device for fieldwork reported here cost approximately €2000 in June 2014. Moving only one platen also makes setting the filament size simpler. Due to the increasing resolution of modern cameras and processing power of laptop computers, the filament midpoint can be easily located by an image analysis code. Aims and scope of the present study The portable rheometer (Seymour) and its use are described in detail. We performed a series of experiments in order to answer the following questions: (i) Does the Seymour device produce comparable results to the conventional laboratory rheometer Trimaster Mk II, for both standardised synthetic liquids and natural pitcher plant fluids? (ii) Does the device yield reliable measurements in the field? (iii) Are there quantifiable differences between the fluids of Nepenthes pitcher plants sampled in greenhouses and in the field? The last two questions were investigated in order to quantify the advantages of a device that allows for on-site measurements of fluid viscoelasticity. Results and discussion Benchmarking studies CaBER™testing requires a rapid initial separation of the platens in order to generate a filament, and negligible movement thereafter. Figure 2 compares the gap separations, expressed in terms of the Hencky strain, measured for the Seymour and the Trimaster for identical stretching settings. The separation speed for both devices is similar, at approximately 75 mm s −1 . Neither device imparts a constant strain rate during the separation stage, while the Seymour produces less overshoot and faster damping than the belt-driven Trimaster. Filament diameter measurements could therefore be collected after 10 ms on the Seymour (17 ms on the Trimaster). This sets a lower limit on the viscosity of liquids that can be measured as the filament stretching should be measured after the platens have stopped moving. Equation (1) predicts that low viscosity liquids will approach filament breakup (D/D 0 approaching zero) at short times. For example, a Newtonian 20 mPa s silicone oil exhibited filament breakup within the 10 ms initial separation period. Figure 3 shows the evolution of filament diameter for the Newtonian silicone oil obtained with the Trimaster and Seymour devices; both series exhibit an essentially linear decrease, as predicted by Equation (1). The dashed lines in this Figure represent the best fit of Equation (1) to the Seymour device’s experimental data using viscosity as an adjustable parameter: for this fitting, X=1 and α=0.0159 N m −1 .The range of reported surface tension values for silicone oil in the literature lie between 0.0159 N m −1 and 0.0213 N m −1 [23]. Viscosity values of 2.37 Pa s, 2.71 Pa s and 3.1 Pa s were t(ms) strain (-) Figure 2 Comparison of linear (Hencky) strain between portable (Seymour) and laboratory (Trimaster) devices. The initial gap, g 0 , was 0.7 mm and reaches a final gap of 1.9 mm. Both devices produced similar separation speed. Seymour produced less overshoot and higher damping than the Trimaster. Collett et al. Plant Methods (2015) 11:16 Page 4 of 16 calculated for initial gap sizes of 0.380 mm, 0.514 mm and 0.612 mm respectively; it is notable that the accuracy of the fit of Equation 1 to these data sets decreased as a function of increasing gap size, with R 2 values of 0.997, 0.989 and 0.982 respectively. The mean value of the viscosity found by these extensional measurements was 2.75 Pa s, some 16% higher than the reported value of 2.37 Pa s. The measurements suggest that smaller initial gap sizes are preferable in order to obtain accurate measurements. All gap sizes used were smaller than the capillary length, lcap ¼ffiffiffiffiffiffiffiffi σ=ρg q,whereρis the fluid density, as recommended by other researchers in this area [24]. The gradients and filament breakup time, t F , increase with initial filament diameter, D 0 , as predicted by Equations (1) and (4), respectively. Figure 4(a) shows excellent agreement between the t F values measured on the two devices at 22°C. The relationship between t F and g 0 also exhibits the trend expected for a Newtonian fluid (Equation (4)), as D 0 is expected to vary with g 0 . Figure 4(b) presents results obtained using the Seymour device at higher temperatures, spanning the range anticipated for field studies (up to 40°C). Filament evolution plots were linear, as in Figure 3, and t F decreases at higher temperature. This is consistent with Equation (4), as η 0 decreases with temperature. Figure 5(a) shows the results obtained for the CMC solution, which is known to be viscoelastic, measured on the Seymour in the laboratory in Cambridge and in the field in Borneo in summer 2014. The filament evolution is presented a function of dimensionless time. The use of dimensionless time accounts for the difference in temperature between the tests in Cambridge and those done in the field; additional insight into this superposition is given by Torres and co-workers [22]. Tests on the Trimaster gave similar results. There is a noticeably sharp transition to filament breakup at t/t F > 0.8, which is not predicted by any of the simple constitutive models (Equations (1)-(3)). The effect of initial gap size on filament break up time is shown in Figure 5(b). The Seymour t f values tended to be shorter than those obtained with the Trimaster. This difference may be related to the protocols: it took longer to load the Trimaster and for the final gap to stabilise, and water evaporation would increase the viscosity and thus filament breakup time. CMC solutions represent complex fluids [25] and these results confirm that the Seymour gives qualitatively similar results to the Trimaster. The above results constitute proof-of-concept of the portable extensional rheometer (Seymour). The data from this device showed good agreement with those obtained with a precision unit, the Cambridge Trimaster Mk II. This was the case for both Newtonian (silicone oil) and complex biopolymer solutions (CMC). Moreover, the Seymour unit is small enough to fit into a climatecontrolled chamber, allowing the effects of temperature and relative air humidity to be studied. The Seymour functions satisfactorily at temperatures up to 40°C, which is essential for field studies in the tropics as well as for medical studies under physiological temperatures. Humidity levels were not investigated as part of this study, but the limit in this regard in field tests is likely to be set by the camera and laptop computer. This broad operational range, together with the small size and weight of the system, renders the Seymour highly suitable for field studies. Application to biological (pitcher plant) fluids Pitcher plant fluids taken from individual N. rafflesiana, N. eymae, and N. maxima obtained from botanical gardens (i.e. greenhouse plants) were tested within one day and periodically thereafter over a period of two weeks. The fresh fluids formed filaments which remained intact for some time (an example for N. maxima in shown in Figure 1(c)), while the N. rafflesiana fluid was not very viscous and the filament often broke before the platens finished moving. We only report data obtained with Seymour here as the Trimaster yielded similar results. Some samples exhibited the formation of satellite droplets, known as ‘beads on a string’(BOAS). The presence of viscoelasticity is a pre-requisite for the formation of BOAS within fluid samples [26] and an example of this behaviour is evident in the field test on N. rafflesiana in the Additional file 1: video. Figure 3 Comparison of filament thinning behaviour for silicone oil. Filament diameters decreased linearly as expected for a Newtonian liquid. Smaller initial gap sizes yielded a better linear fit. Data sets obtained with Seymour (S, open symbols) and Trimaster (T, solid symbols) devices showed strong agreement. Data are decimated for clarity. Collett et al. Plant Methods (2015) 11:16 Page 5 of 16 The regression coefficients quantifying the fit of Equations (1), (2) and (3) to filament thinning profiles (plots of D/D 0 or ln(D/D 0 )againstt)aregiveninTable1. The plots in Figure 6, of ln(D/D 0 ) against time, suggested by Equation (2), show an approximately linear trend for all three fluids. This indicates that the viscoelasticity is adequately described by the simple, single parameter UCM model. The values of the relaxation time, Figure 4 Filament break-up times for silicone oil (Newtonian fluid). (a) Measurements obtained with Trimaster Mk II and Seymour at 22°C show excellent agreement. (b) Higher temperatures lead to shorter break-up times (data obtained with Seymour device). Collett et al. Plant Methods (2015) 11:16 Page 6 of 16 λ UCM , extracted from model fitting are reported in Table 1. The N. rafflesiana value, of 3 ms, is small and could not be measured reliably with either of the Trimaster or Seymour devices. The N. eymae,andN. maxima values aremorethananorderofmagnitudesmallerthanrelaxation times of ~ 1 s reported by Gaume and Forterre for N. rafflesiana [9], confirming the desirability to perform tests in the field if possible. Effect of sample storage on pitcher plant viscoelasticity It has been observed that pitcher plant fluids stored for over one month lose their stickiness (Bauer, unpublished), Figure 5 CMC solution filament thinning behaviour showing complex behaviour. (a) Seymour testing, alongside best fit lines for the Newtonian and Giesekus models. The data are plotted against dimensionless time, t/t F . Laboratory and field measurements largely agree. (b) Filament break-up times increased linearly with initial gap size. Seymour yielded consistently shorter break-up times than the Trimaster. Collett et al. Plant Methods (2015) 11:16 Page 7 of 16 which is an indicator of a reduction in viscoelasticity. This was reproduced in laboratory studies on (greenhouse-sourced) fluid samples of N. maxima and N. eymae. Fluids were found to lose their viscoelastic properties when stored at ambient temperatures over 2–4 weeks. The samples were stored in sealed containers at room temperature and small aliquots were extracted for testing on the Seymour device at different times over two weeks and a month. The testing period was longer for N. eymae as the initial t f value was larger and quantitative data could be obtained over a month. Figure 7 shows noticeable differences in viscoelasticity with storage time. These data were fitted to all three expressions to quantify the change in viscoelastic behaviour. The R 2 values are reported in Additional file 2: Table S1. The plots show an approximately linear decrease in ln(D/D 0 ) with time, indicating that the viscoelasticity is adequately described by the simple, single parameter, UCM expression and this model provided a better description for most cases for both fluids across the sample sets. The Giesekus model gave comparable R 2 values but the low (sometimes zero) magnitude of η o cast doubt on the validity of the results. The quality of fit of the Newtonian model for N. eymae improved considerably with storage time. This trend indicates that the fluids are losing their viscoelastic properties with time when stored at ambient temperature. Figure 7 also shows a noticeable reduction in t f with storage time, which is again consistent with the fluids changing from viscoelastic to Newtonian behaviour. The relaxation times extracted from model fitting are reported in Table 2 and also decrease over the storage period, by over an order of magnitude. This will give rise to predominantly Newtonian behaviour, which can be illustrated using the result for the Giesekus fluid in Equation (3). If the product of the terms is small compared to the non-dimensional filament diameter, D/D 0 , then it can be shown that 4a−3ðÞλGln D=D0 ðÞþ 2η0 αD0−DðÞ¼tð5Þ As λ G decreases, the viscoelastic contribution (first term on the left hand side) becomes negligible and viscous behaviour dominates. Figure 8 shows how λ UCM changes over the storage period. The almost linear trend for the N. eymae fluid on this log-linear plot suggests a first order decay. Elucidating this behaviour requires further work and analysis of the biopolymer components. The above results confirmed and quantified the previously observed decay of viscoelasticity for pitcher fluids in storage. Both N. maxima and N. eymae fluids showed a tendency to become more Newtonian over the course of two to four weeks (Figure 7). The time–dependent reduction in the relaxation time of longer-stored fluid samples provided a second quantitative measure of the loss of fluid viscoelasticity (Figure 8). The two fluids considered here were sampled from newly opened pitchers so the decay is unlikely to be caused by environmental factors or by the interaction with captured prey or pitcher-colonising infauna organisms. Some insight into this behaviour was provided by storing a sample under chilled conditions, at 4°C. An aliquot was withdrawn and allowed to warm to room temperature for testing. Chilling effectively halted the change in viscoelasticity, as shown for N. maxima in Additional file 3: Figure S1, which may be related to inactivation of enzymes: this is the subject of ongoing work. The Table 1 Parameter estimates and goodness of fit for different fluid models for pitcher fluids from three Nepenthes species (samples obtained from greenhouse plants, measurements performed with Seymour) Species Equation (1) Equation (2) Equation (3) η 0 R 2 λ UCM R 2 η 0 aλ G R 2 (Pa s) (ms) (Pa s) (−) (ms) N. rafflesiana 3.47 0.964 2.95 0.994 1.97 0 1.79 0.995 N. eymae 26.7 0.503 29.8 0.958 0.0644 0 32.4 0.979 N. maxima 20.6 0.599 20.6 0.991 0.0491 0 21.7 0.994 Figure 6 Filament evolution profiles for fresh greenhousesampled of pitcher fluids. N. eymae (diamonds) and N. maxima (triangles) both showed clearly viscoelastic behaviour while the N. rafflesiana filament broke before the pistons finished separating. The UCM model (dashed lines) provided the best fit for all three samples; λ UCM values given in Table 1. Experimental data have been decimated for clarity. Collett et al. Plant Methods (2015) 11:16 Page 8 of 16 measure (i) a silicone oil (Newtonian liquid), and (ii)solutions of carboxymethylcellulose (CMC), a biopolymer exhibiting non-Newtonian behaviour in solution. The silicone oil (Silicone fluid f191/1300, batch no. 805, Ambersil Ltd, UK) had a viscosity of 2.37 Pa s. Aqueous solutions of 2 wt% carboxymethylcellulose (molecular weight approximately 750 kDa, BDH Chemicals, UK) were prepared by gentle stirring for 4 h. CMC solutions are viscoelastic shear-thinning fluids and are relatively stable [25]. The shear viscosity of the silicone oil and CMC solutions was measured on a Bohlin CVO 120 HR controlled stress rheometer using 25 mm diameter, smooth parallel plates with a 0.5 mm gap at 22°C. The shear rates studied ranged from 0.1 to 3000 s −1 . The volume of fluid required for these tests (250 μL) was considerably larger than that needed for the Seymour tests (approximately 1 μL). The CMC solution exhibited shear thinning with a zero shear rate viscosity, η 0 , of 2.95 Pa s, similar to the viscosity of the silicone oil. Botanical garden pitcher plant studies Samples of pitcher plant fluid for N. maxima and N. alata were obtained from individual plants at the Cambridge Botanic Gardens, while N. rafflesiana and N. eymae fluids were obtained from Kew Gardens, London. Greenhouse relative humidity levels were maintained between 34% and 92%. Freshly opened pitchers were chosen where possible. Ideally, fluid would be extracted from an unopened pitcher using a syringe, but in many cases the pitchers had already opened, and there was some contamination by captured flies and plant detritus in the fluid. Approximately 5 ml of liquid was removed and stored in a small sterilised Nalgene bottle. Samples were stored at room temperature (around 22°C), and tested repeatedly over a period of 37 days (N. eymae)/16 days (N. maxima). Field trials Field measurements were performed in Brunei Darussalam, NW Borneo, between July and September 2014. The test fluids used for the benchmarking studies (silicone oil, CMC) were used to calibrate the measurements in the field. Fluid samples (100 μL per sample) were taken from just opening N. rafflesiana pitchers using a micropipette and were either measured immediately on site (Figure 12, see also the Additional file 1: video), or transferred to 2 mL sterile screw-top vials and transported back to a nearby field station. Within a maximum of 4 h after sampling, these samples were measured in a room at temperatures between 24°C and 27°C. The pitcher plant fluid samples were then stored at room temperature (22-30°C) and re-measured after 20 days. Storage at room temperature was chosen to mimic conditions during expeditions or at remote field stations where reliable refrigeration may not be available. Confirmation of model assumptions Discussion of the characteristic timescales for CaBER™ experiments can be found in the literature [8,9,31]; these give indications of when filament stretching is the controlling mechanism. The inertial (Rayleigh) time scales for the tests presented here, given by √(ρD 0 3 /8α), ranged from 0.02-0.04 ms whereas the viscous times, (given by η 0 D 0 /2α), lay between 2 and 4 ms. The majority of tests were then considerably longer than both these times. The elastocapillary number, comparing the relaxation time in extension to the viscous time, Ec=2λα/η 0 D 0 , ranged from 9 for the greenhouse N. maxima tests to 2100 for the wild N. rafflesiana (Figure 10). These data confirm the presence of viscoelasticity. Additional files Additional file 1: Video Operation of Seymour in the field, measuring a sample of N. rafflesiana pitcher fluid in the natural habitat of the plant in Brunei Darussalam, Borneo. Additional file 2: Table S1. Effect of storage at room temperature on the goodness of fit (quantified by the correlation coefficient, R 2 ), for N. eymae and N. maxima fluid obtained from greenhouses (one pitcher for each species). Additional file 3: Figure S1. Effect of storage under chilled conditions. Filament thinning behaviour for greenhouse-sourced N. maxima pitcher fluid. Sample tested at Day 1 then stored at 4°C for up to 13 days, for comparison with Figure 7(b). Each aliquot was brought to room temperature before testing. Data decimated for clarity. Additional file 4: Table S2. Fitted model parameters for 11 newly-opened pitchers of N. rafflesiana growninthefield. Additional file 5: Table S3. Fitted model parameters for 9 of the 11 pitchers of N. rafflesiana in Additional file 4: Table S2 following storage for 20 days at 25-30°C. Two experimental measurements were made for each pitcher. Samples 13b and 23 were not tested. Additional file 6: Figure S2: Frames from a typical image sequence. The figure shows the original and processed images at the start of a typical filament thinning experiment, two midpoint filament thinning times and at a time close to filament breakup. Abbreviations BOAS: Beads on a string; CMC: Carboxymethylcellulose; UCM: Upper Convected Maxwell (rheological model). Competing interests The authors declare that they have no competing interests. Authors’contributions CC, AA and EC all participated in the design and commissioning of the device, laboratory experimental studies, and reading the manuscript. GC and LP devised, designed and constructed the device. LT-P assisted with experimental studies and rheology work. UB performed the experimental work in Brunei and helped to draft the manuscript. BH and DIW conceived of the study, co-ordinated the work and drafted the manuscript. BH wrote the software tools for image analysis. All authors read and approved the final manuscript. Authors’information UB is an early career research fellow in the School of Biological Sciences at the University of Bristol, UK, working on the biomechanics and functional morphology of pitcher plants. All the other authors were based in the Department of Chemical Engineering and Biotechnology at Cambridge, UK, at the time of this study. CC, AA and EC performed this work as final year/diploma research projects under the direction of chemical engineering academics BH and DIW. GC and LP are assistant staff in the Department’s workshops. Collett et al. Plant Methods (2015) 11:16 Page 15 of 16 Acknowledgements This work grew from a conversation over dinner at Jesus College, Cambridge between UB, DIW, Dr Walter Federle (Department of Zoology, Cambridge) and Prof. Francis Gadala-Maria (Department of Chemical Engineering, University of South Carolina). Additional assistance and advice from the following is gratefully acknowledged: Dr Simon Butler, John Gannon and Kevin Swann (Department of Chemical Engineering & Biotechnology, Cambridge), Alex Summers (Cambridge University Botanic Garden), and Dr Ulmar Grafe (Faculty of Science, Universiti Brunei Darussalam). The image processing tool in Fiji was based on the code kindly provided by Dr Damian Vadillo. Mathias Scharmann (Institute of Integrative Biology, ETH Zürich) kindly took the video showing the experimental protocol. The following financial support is gratefully acknowledged: a Henslow Research Fellowship from the Cambridge Philosophical Society and a Leverhulme Early Career Fellowship for UB; a visiting research fellowship (POS-A/2012/116) for MDT from Xunta de Galicia’s Consellería de Cultura, Educación e OrdenaciónUniversitaria of Spain and the European Union’s European Social Fund; and a summer project grant for CC from Sidney Sussex College, Cambridge. The fare at the dinner was also most agreeable. 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