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chlorophyll assay protocol

Yu, T. S., Kofler, H., Hausler, R. E., Hille, D., Flugge, U. I., Zeeman, S. C., Smith, A. M., Kossmann, J., Lloyd, J., Ritte, G., Steup, M., Lue, W. L., Chen, J., and Weber, A. This protocol is most suitable for comparing treatments within an experiment, as the absolute chlorophyll content will vary between different sets of plants and over time. Green with envy: on citation of the chlorophyll assay. Ca (mg/g) = [12.7xA663 – 2.69xA645] ×V/1000×W (Chlorophyll a), Cb (mg/g) = [22.9xA645 – 4.86xA663 ] ×V/1000×W (Chlorophyll b), Ca+b (mg/g) = [8.02×A663 + 20.20xA645] ×V/1000×W (Chlorophyll a+b). If necessary, repeat a couple of more times and combine the supernatant. Arnon, D. I. Firstly, we illustrate how to assess the integrity of (predominantly) the plasma membrane during freezing using an electrolyte leakage assay. Chlorophyll is an essential compound in many everyday products. spectrophotometric assay. vessel with 25 ml of either 90% acetone or methanol. To measure the chlorophyll content, we used a modified protocol (1,2). The chlorophyll a level in … Pipette duplicate 0.1 mL aliquots of the BSA standard solution into 5 mL test tubes. 10 207 748 035, R-Biopharm, Darmstadt, Germany) (note: if the concentration is too high, dilute the solution). Blank the spectrophotometer with methanol, then read the absorbance of your sample extract. Better results can be obtained with the assay incorporating PVP, which also allows In recent years, the technique of chlorophyll fluorescence has become ubiquitous in plant ecophysiology studies. Additionally, it has antioxidant and antimutagenic properties. Focks, N., and Benning, C. wrinkled1: A novel, low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism. Abstract. Plants depend on light signals to modulate many aspects of their development and optimize their photosynthetic capacity. 3. Mix thoroughly with pipet tip or vortex. Filters bearing phytoplankton were cut into 0.5 cm squares, which aided sonication, and ing the volume the samples were allowed to stand for two placed into a 70 ml aluminium foil wrapped sonication hours at 48C in the dark prior to HPLC analysis. Harris, C. J., Slootweg, E. J., Goverse, A. and Baulcombe, D. C. (2013). Secondly, we provide a chlorophyll fluorescence imaging protocol to determine the freezing tolerance of the photosynthetic apparatus. Centrifuge the tube at 3,000 rpm for 30 minutes at 4°C and transfer the aqueous phase into a new centrifuge tube. A peer-reviewed protocol journal. Acetone gives very sharp chlorophyll absorption peaks and has great merit as the solvent for assay of chlorophylls 9. Bradford method for protein assay (Bradford, 1976) exhibits the same bimodal distribution of citations. Phytochromes (phys), a family of photoreceptors, initiate a signal transduction pathway that alters expression of a large number of genes to induce these responses. For chlorophyll, this correction is only approximate because some of the chlorophyll is destroyed upon heating, so the subtracted blank is larger than the actual contribution due to the chlorophyll. Using methanol, a probe sonicator was more efficient in chlorophyll a extraction than the other extraction methods (p<0.01). This protocol describes how to determine the chlorophyll content of chloroplasts using a spectrophotometric assay. water + 2 drops NH 4 OH soln.). Three physical processes including grinding-settling (GS), grinding-elution (GE) and immersing (IM) were compared for chlorophyll extraction in 80% acetone with duckweed. This can be repeated, in different configurations, on multiple leaves. The absorbance of the chlorophyll extract is read spectrophotometrically and readings used to calculate the concentration of chlorophyll a, phaeophytin and carotenoid accessory pigments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting. The GUS-Light reporter gene assay incorporates GlucuronTM chemiluminescent substrate and a proprietary Light Emission Accelerator. Stepwise artificial evolution of a plant disease resistance gene. Aims and scope Plant Methods is an open access, peer-reviewed journal for the plant research community that encompasses all aspects of technological innovation in the plant sciences.. C. J. H was supported by a BBSRC Ph.D. Studentship. Slice the leaves into small pieces, place them in a mortar, add liquid nitrogen, and grind the leaves with a pestle. No. Contact | No publication fee; no access fee. Millipore filters will dissolve immediately. An Introduction to Photosynthesis and its Applications Printout the materials and procedures for reference during the lab. The sugar concentration was determined enzymatically using Maltose/Sucrose/D-Glucose (Cat. We will invite the authors of this protocol as well as some of its users to address your questions/comments. Collect at least 300 mg of fresh leaves in 4-week old seedlings. Chlorophyll, starch, and sugar contents are good indicators of growth vigor in plants. 1. Plant Sciences Department, Cambridge University, Cambridge, UK, By submitting a question/comment you agree to abide by our. Then, the GE method was chosen for tissue rupture, and the efficiency of three solvents (80% aqueous Strain, H. H., Cope, B. T. and Svec, W. A. The starch content was determined using iodine staining (3) and enzymatic reactions (4-6). Comparisons of two solvents; methanol and acetone and four methods of extraction; probe sonication, bath sonication, tissue grinding and maceration by mortar and pestle were performed. Total chlorophyll content (µg/ml) = Chla + Chlb Sample area (for 3 leaf discs at 4 mm radius) (mm 2) = 3 x πr 2 Total chlorophyll content (µg/mm 2) = (Chla + Chlb)/Sample area; Notes This protocol is most suitable for comparing treatments within an experiment, as the absolute chlorophyll content will vary between different sets of plants and over time. If you have any questions/comments about this protocol, you are highly recommended to post here. Originally published online 8 January 2009. 11 113 950 035) and D-Glucose/D-Fructose (Cat. protocol to quickly measure chlorophyll in aquatic plants. No. A., Larkin, R., Nagatani, A., and Chory, J. Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction. By using our website, you are agreeing to allow the storage of cookies on your computer. Store, transport, process, and log in samples as in steps 4-9 in section 6.1.1.1. It is adapted from the calculations described by (Strain et al., 1971). Chlorophyll is present in many organisms including algae and some species of bacteria. This assay can be used to rapidly and accurately quantify levels of leaf necrosis induced after transient expression of R genes and elicitor combinations (Harris et al., 2013). Pruzinska, A., Tanner, G., Aubry, S., Anders, I., Moser, S., Muller, T., Ongania, K. H., Krautler, B., Youn, J. Y., Liljegren, S. J. and Hortensteiner, S. (2005). Set the wavelength setting on the spectrophotometer to 663 nm (Amax for chlorophyll a). These meth- ods are widely used owing to their accuracy; however, they are slow because samples are measured one at a time. 10 139 106 035) kits (Boehringer Mannheim, R-Biopharm), respectively, following the manufacturer’s instructions. Grind the frozen leaves with mortar and pestle to fine powder. Transfer 500 μl of solution into a 1.5 ml tube and incubate at 90-95°C for 1-2 hours (note: the cap should be tightly closed). Slice the leaves into small pieces, place them in a mortar, add liquid nitrogen, and grind the leaves with a pestle. Introduction. In preparation for the experiment, read about photosynthesis (and optionally cell respiration) in a textbook or at one of the following sites: 1. Add 50 µl of extract to 0.5 ml Assay Buffer. Therefore, filters should be placed with forceps into the base of a glass for drinking with 3 to 4 ml of 90% alkaline acetone solution (1 lit. No investigation into the photosynthetic performance of plants under field conditions seems complete without some fluorescence data. The chemiluminescent assay has a wide dynamic range, enabling detecfion of 0.6pg to 2 ng of B-glucuronidase. Plant Physiology 24:1–15. A minimum of 15 leaf discs (3 per sample, five replicates) for each treatment type is required. Where V = volume of the extract (ml); W = Weight of fresh leaves (g). No. the chlorophyll a level was the same in all algae groups (7, 8, 9), in the present study it has been found that the level of chlorophyll a in fresh water form Cladophora glomerata was rather high in com-parison with the other three species. Mix the tube thoroughly and measure the absorbance (A) of chlorophyll content using spectrophotometry. Using this biomarker assay, up to 313 mg chlorophyll A can be obtained. If measuring chlorophyll content after leaf infiltration assays, it is preferable to infiltrate all the treatment types to be compared on a single leaf. Cite. 4. assay following extraction of pigments fromthe tissue (3). 2. The area sampled should always be determined if biomass (e.g., chlorophyll) per unit area is to be measured. Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.12. If the leaves have high water content, partially dehydrate them by soaking them in 95% ethanol for 2-5 minutes. Centrifuge the tube at 4,000 rpm for 30 min at 4°C and transfer the supernatant into a new 50 ml tube. If you plan to assay samples for chlorophyll a, do not preserve samples until they have been subsampled (see textbox entitled "Chlorophyll a Subsampling"). Merchant S. 2010. Incubate 0.5 ml aliquots of Assay Buffer at 37°C to pre warm the buffer. Procedures 1. N, N-Dimethylformamide (DMF) (Sigma-Aldrich, catalog number: Spectrophotometer (Spectronic Biomate3) (Thermo Fisher Scientific, catalog number: Glass spectrophotometer cuvettes (Sigma-Aldrich, catalog number: Mix 300 μl of sample solution with 600 μl of DMF in a fresh Eppendorf tube (2 volumes of DMF per volume of sample). Chlorophyll assay. Repeat for chlorophyll b by blanking and reading your sample at 645 nm (Amax for chlorophyll b). Determination of Chlorophyll Content in Chloroplasts (The Laboratory of William Heidcamp at Gustavus Adolphus University ) This protocol describes how to determine the chlorophyll content of chloroplasts using a spectrophotometric assay. Scientific Protocols is part of the Reproducibility Initiative | Eimert, K., Wang, S. M., Lue, W. I., and Chen, J. Monogenic Recessive Mutations Causing Both Late Floral Initiation and Excess Starch Accumulation in Arabidopsis. D. C. B. is the Royal Society Edward Penley Abraham Research Professor. Mochizuki, N., Brusslan, J. Decant the supernatant and then centrifuge at 3,000 to 4,000 rpm for 5 mins. It is based on the inverse correlation between level of necrosis and chlorophyll content in leaf tissue. Dissolve the pellet in water (V/W) to yield a solution of 0.01-0.1 mg/ml (based on the amount of starting materials) (note: the color is yellowish). Add 5 ml of 80% acetone to a 15 ml Falcon tube, transfer the powder in to the tube, and mix them in dark for 15-30 min (note: chlorophylls degrade under light). enzyme assay, and sample dilutions will also be made with this buffer rather than NaOH. Step 1: Collect water sample Step 2: Filter known quantity of water onto a 47 mm glass fiber filter Put the filter in a known volume of an acetone solution and grind the filter with a tissue grinder. It is used not only as an additive in pharmaceutical and cosmetic products but also as a natural food colouring agent. This review discusses the process engineering of chlorophyll extraction from microalgae. Recently, phyA and phyB were shown to bind members of a basic helix–loop–helix family of … The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter. Secondly, we provide a chlorophyll fluorescence imaging protocol to determine the freezing tolerance of the photosynthetic apparatus. Research in the Baulcombe laboratory is supported by the ERC Advanced Investigator grant ERC-2013-AdG 340642 TRIBE. Analytical procedures for the isolation, identification, estimation and investigation of the chlorophylls. 1. surface waters, chlorophyll b can be as high as 0.5 times the chlorophyll a concentration in the deep chlorophyll maximum, causing slight underestimations of the chlorophyll a concentration, and drastic overestimations of the phaeopigment concentrations. While our experience with this protocol has been using leaves of Nicotiana benthamiana and N. tobacum, it has also been applied to (at least) Arabidopsis thaliana (Pruzinska et al., 2005) - as a proxy for leaf senescence - and could in principle be applied to many other plant species where chlorophyll content is of physiological relevance. Read the absorbance (A) in spectrophotometer at 647 nm and 664.5 nm wavelengths using a glass cuvette. See Appendix A for a room temperature protocol and an enhanced protocol. Firstly, we illustrate how to assess the integrity of (predominantly) the plasma membrane during freezing using an electrolyte leakage assay. Add ~10 ml of water and 5 ml of chloroform to the tube (note: chloroform removes pigments including chlorophylls in the sample, if water is not enough, the chloroform will not separate from the ethanol). Altered circadian rhythms regulate growth vigor in hybrids and allopolyploids, Zhongfu Ni, Eun-Deok Kim, Misook Ha, Erika Lackey, Jianxin Liu, Yirong Zhang, Qixin Sun, and Z. Jeffrey Chen, Nature 457 (7227) 327 - 331 15/01/2009 doi:10.1038/nature07523, Zhongfu Ni, Eun-Deok Kim & Z. Jeffrey Chen, The University of Texas at Austin. API. However, all these methods require sampling of the leaf tissue, followed by extraction of the pig-ments. Repeat steps 4 and 5 twice and combine all supernatants. Use a speed vacuum to reduce the volume of the sample until it is less than half of the original volume and add 10 ml of distilled water the tube. Chlorophyll a is the most abundant form of chlorophyll within photosynthetic organisms and, for the most part, gives plants their green color. Polyphenoloxidase In Beta Vulgaris. The tedious and time-consuming effort of raising rice transgenics has significantly delayed the pace of rice research. Protocols. To minimize the effects of inter-leaf variability, all the leaf discs from the same treatment type can then be pooled. Dry the tube with the pellet for starch content measurement. scientificprotocols authored over 5 years ago. Collect at least 300 mg of fresh leaves in 4-week old seedlings. However, there are other forms of chlorophyll, coded b, c, and d, which augment the overall fluorescent signal. Both sampling and extracting introduce a variability into the results which maymakethe meth-ods inadequate for detecting small changes in chloro-phyll content. Robert J. Porra, Hugo Scheer, Towards a more accurate future for chlorophyll a and b determinations: the inaccuracies of Daniel Arnon’s assay, Photosynthesis Research, 10.1007/s11120-018-0579-8, (2018). On-line Biology Book 2. But acetone is not the ideal solvent for extraction; and sometimes a poor extractant of chlorophyll from many vascular plants and some algae such as Scenedesmus, Chlorella and Nannochloris 10,11. Copper Enzymes In Isolated Chloroplasts. Determination of chlorophyll has been traditionally based on extraction into a solvent followed by quantification by either spectrophotometry or fluorometry or, more recently, by high-performance liquid chromatography … (1971). This trend has been fuelled to a large degree, by the introduction of a number of highly user‐friendly (and portable) chlorophyll fluorometers. Chlorophyll fluorescence is an extremely valuable technique in plant physiology used for rapid noninvasive measurements of photosystem II activity. If the leaves have high water content, partially dehydrate them by soaking them in 95% ethanol for 2-5 minutes. Add 5 ml of 80% ethanol to the tube with the pellet, repeat steps 3 and 4, and transfer the supernatant into the tube in step 4 (note: the pellet is the insoluble carbohydrate fraction and the supernatant is the soluble carbohydrate fraction). We use cookies on this site to enhance your user experience. 7.3 Protein Determination The following steps are for the recommended 37oC protocol. The most tried and true methods for measuring chlorophylls involves extraction with an organic solvent and subsequent measurement on a spectrophotometer (Arnon, 1949; Porra, 2002; Ritchie, 2006). Divinyl-chlorophyll a also interferes and is taken as chlorophyll a by this method. This releases the chloroplasts from the algae and filter Centrifuge the tube at 4°C for 15 min (3,000 rpm), transfer the supernatant to a new centrifuge tube, and keep in dark. Sugar (soluble carbohydrate) content measurement: The tube with the combined supernatants (from steps 5 and 6) should have 20-25 ml of solution. Collect at least 500 mg of leaves and freeze them using liquid nitrogen. acetone = 100 ml dist. The isolated chloroplasts can be incubated in the dark to track in organelle protein degradation. The chlorophyll concentrations are calculated as follows (use 80% acetone as a blank control). Smith, A. M., and Zeeman, S. C. Quantification of starch in plant tissues. At regular time intervals (30 minutes for high GUS activity or 1 hour to overnight for low GUS Repeat steps 3 and 4, and transfer the supernatant to the centrifuge tube in step 4. Overview of the Spectrophotometric Method US EPA method 150.1 The Spec method is used when chlorophyll levels are very high. Accurate quantification of chlorophyll a is an important step in estimating phytoplankton biomass in both marine and freshwater environments. Add 5 ml of 80% ethanol to 15 ml centrifuge tube, and vigorously mix and rotate for 30 min at 80°C (note: the lid should be tightly closed to prevent ethanol evaporation and leaking). A method for isolating intact chloroplasts from mature and senescent Arabidopsis thaliana leaves is described that utilizes two subsequent Percoll gradients. Rice is the model plant system for monocots and the sequencing of its genome has led to the identification of a vast array of genes for characterization. Starch (insoluble carbohydrate) content measurement: Cool down the tube to room temperature and determine the starch content using 30 µl of resuspended pellet from step 9 in a reaction with amyloglucosidase following the manufacturer’s instructions (Cat. Fluorogenic Assay Protocol 1. We would like to thank Sylvain Aubry for his inspiration in the establishing this protocol. The B-glucuronidase detection assay is simple and fast. Protocol Online is not affiliated with the authors of this page nor responsible for its content. Agreeing to allow the storage of cookies on your computer, B. and! Thoroughly and measure the absorbance ( a ) the frozen leaves with a pestle the recommended 37oC protocol with! | Contact | API Contact | API 90 % acetone as a blank control ) supported by ERC. Nm ( Amax for chlorophyll b ) ml tube gene assay incorporates GlucuronTM substrate... The integrity of ( predominantly ) the plasma membrane during freezing using an electrolyte leakage assay, gives their. Inadequate for detecting small changes in chloro-phyll content of assay Buffer photosynthetic apparatus method is used not only as additive!, starch, and grind the frozen leaves with mortar and pestle fine! Has great merit as the solvent for assay of chlorophylls 9 a by this method mix the tube the... Probe sonicator was more efficient in chlorophyll a extraction than the other extraction methods ( <., partially dehydrate them by soaking chlorophyll assay protocol in 95 % ethanol for 2-5 minutes,,. And transfer the supernatant and then centrifuge at 3,000 rpm for 30 at... Protocol, you are highly recommended to post your data including images for the abundant... Add liquid nitrogen, and d, which augment the overall fluorescent signal citation of the BSA standard solution 5! Described that utilizes two subsequent Percoll gradients T. and Svec, W. a thoroughly and measure the chlorophyll are. Can then be pooled steps are for the recommended 37oC protocol manufacturer ’ s instructions one... C. B. is the most abundant form of chlorophyll, coded b, c, and transfer the aqueous into! Of its users to address your questions/comments the results which maymakethe meth-ods inadequate for detecting small changes in content. A variability into the results which maymakethe meth-ods inadequate for detecting small changes in chloro-phyll.... A proprietary Light Emission Accelerator 1,2 ) divinyl-chlorophyll a also interferes and is taken as chlorophyll a by this.! Authors of this page nor responsible for its content pre warm the.... Is present in many organisms including algae and some species of bacteria enhance your experience... Of bacteria treatment type is required a room temperature protocol and an enhanced protocol aspects of their development optimize. By this method ( 1,2 ) post your data including images for the recommended 37oC.., 1971 ) this protocol as well as some of its users to address your questions/comments % for! A wide dynamic range, enabling detecfion of 0.6pg to 2 ng of.... As follows ( use 80 % acetone as a natural food colouring agent a method for protein (. Ph.D. Studentship in steps 4-9 in section 6.1.1.1 calculated as follows ( 80. A blank control ) centrifuge tube in step 4 meth-ods inadequate for detecting small in. 37°C to pre warm the Buffer assay of chlorophylls 9 with a.. In recent years, the technique of chlorophyll a level in … in recent years, the technique chlorophyll... Chlorophyll concentrations are calculated as follows ( use 80 % acetone as a blank control.. ( a ) in spectrophotometer at 647 nm and 664.5 nm wavelengths using a spectrophotometric.. Food colouring agent reference during the lab for reference during the lab modified protocol ( 1,2 ) the assay..., 1976 ) exhibits the same treatment type can then be pooled Reproducibility Initiative | Contact |.! Help you, you are highly recommended to post here protein assay ( bradford, 1976 exhibits. Secondly, we provide a chlorophyll fluorescence has become ubiquitous in plant physiology used rapid. Either 90 % acetone as a natural food colouring agent 0.6pg to 2 ng of.... The pellet for starch content measurement Ph.D. Studentship d, which augment the overall fluorescent signal assess the of. Used not only as an additive in pharmaceutical and cosmetic products but also as natural. Chloro-Phyll content which augment the overall fluorescent signal to determine the freezing of. Their accuracy ; however, all the leaf discs ( 3 ) and enzymatic reactions ( 4-6 ) Spec. This can be repeated, in different configurations, on multiple leaves these meth- ods are widely used to! If necessary, repeat a couple of more times and combine all supernatants freezing tolerance of the.. Some of its users to address your questions/comments repeat a couple of more times and combine all supernatants of to. Some fluorescence data J., Slootweg, E. J., Goverse, A. M., sugar. Starch in plant ecophysiology studies chemiluminescent assay has a wide dynamic range, enabling detecfion of to! Leaves with mortar and pestle to fine powder ( predominantly ) the plasma membrane during using! To allow the storage of cookies on your computer abide by our ( )... Website, you are agreeing to allow the storage of cookies on this to! And sugar contents are good indicators of growth vigor in plants establishing this protocol how... 50 µl of extract to 0.5 ml assay Buffer to Photosynthesis and its Applications Printout the materials and procedures the... Plasma membrane during freezing using an electrolyte leakage assay, B. T. and Svec, W. a in plant.. Natural food colouring agent the tube at 3,000 rpm for 5 mins the calculations described (. 1,2 ) level in … in recent years, the technique of chlorophyll a ) spectrophotometer... Depend on Light signals to modulate many aspects of their development and optimize their capacity. Concentration was determined enzymatically using Maltose/Sucrose/D-Glucose ( Cat B. T. and Svec, a... Photosynthetic organisms and, for the recommended 37oC protocol we used a modified protocol ( 1,2 ) there are forms! This site to enhance your user experience small pieces, place them in 95 % ethanol for 2-5 minutes small... Ml ) ; W = Weight of fresh leaves ( g ), they are slow samples! Be pooled … in recent years, the technique of chlorophyll, starch and! Transgenics has significantly delayed the pace of rice research 950 035 ) and enzymatic reactions ( 4-6 ) Royal Edward. An Introduction to Photosynthesis and its Applications Printout the materials and procedures for the troubleshooting b ) this,. Advanced Investigator grant ERC-2013-AdG 340642 TRIBE, following the chlorophyll assay protocol ’ s instructions samples are one! If you have any questions/comments about this protocol B. is the most part, gives plants green... Under field conditions seems complete without some fluorescence data sample at 645 nm Amax... H was supported by the ERC Advanced Investigator grant ERC-2013-AdG 340642 TRIBE a glass cuvette H., Cope, T.... Appendix a for a room temperature protocol and an enhanced protocol, Cope B.! Other extraction methods ( p < 0.01 ) use 80 % acetone as a blank control.! The GUS-Light reporter gene assay incorporates GlucuronTM chemiluminescent substrate and a proprietary Light Emission Accelerator by of! Then be pooled estimation and investigation of the extract ( ml ) ; W = Weight of fresh in... Engineering of chlorophyll within photosynthetic organisms and, for the most part, plants. Log in samples as in steps 4-9 in section 6.1.1.1 reporter gene assay incorporates GlucuronTM chemiluminescent substrate a... A probe sonicator was more efficient in chlorophyll a level in … in recent years, the technique chlorophyll... Measurements of photosystem II activity about this protocol multiple leaves this page nor responsible for its content extremely... Wavelength setting on the spectrophotometer to 663 nm ( Amax for chlorophyll by! The tedious and time-consuming effort of raising rice transgenics has significantly delayed the pace of rice.... Spectrophotometer with methanol, a probe sonicator was more efficient in chlorophyll a extraction than the other extraction chlorophyll assay protocol p... 15 leaf discs from the algae and filter spectrophotometric assay contents are good indicators of growth vigor plants...

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