参见作者原研究论文

本实验方案简略版
Dec 2017

本文章节


 

Determination of Storage (Starch/Glycogen) and Total Saccharides Content in Algae and Cyanobacteria by a Phenol-Sulfuric Acid Method
苯酚-硫酸法测定藻类和蓝藻中淀粉/糖原的储存和总糖含量   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

This is a protocol for quantitative determination of storage and total carbohydrates in algae and cyanobacteria. The protocol is simple, fast and sensitive and it requires only few standard chemicals. Great advantage of this protocol is that both storage and total saccharides can be determined in the cellular pellets that were already used for chlorophyll and carotenoids quantification. Since it is recommended to perform the pigments measurement in triplicates, each pigment analysis can generate samples for both total saccharide and glycogen/starch content quantification.

The protocol was applied for quantification of both storage and total carbohydrates in cyanobacteria Synechocystis sp. PCC 6803, Cyanothece sp. ATCC 51142 and Cyanobacterium sp. IPPAS B-1200. It was also applied for estimation of storage polysaccharides in Galdieria (IPPAS P-500, IPPAS P-507, IPPAS P-508, IPPAS P-513), Cyanidium caldarium IPPAS P-510, in green algae Chlorella sp. IPPAS C-1 and C-1210, Parachlorella kessleri IPPAS C-9, Nannochloris sp. C-1509, Coelastrella sp. IPPAS H-626, Haematococcus sp. IPPAS H-629 and H-239, and in Eustigmatos sp. IPPAS H-242 and IPPAS C-70.

Keywords: Sugars (糖), Carbohydrates (碳水化合物), Polysaccharides (多糖), Colorimetry (比色法), Spectrophotometry (分光光度法), Synechocystis (蓝藻), Chlorella (小球藻), Haematococcus (红球藻)

Background

Carbohydrates play a number of roles in the metabolism of algae and cyanobacteria. As nicely summarized by Raven and Beardall (Raven and Beardall, 2003), carbohydrates represent major sink of intermediates in carbon reduction/oxidation pathways (photosynthesis and photorespiration), they provide skeletons required for growth (e.g., for amino-acid or cell wall biosynthesis), they represent sources of ATP and reducing equivalents (through respiration pathways), they serve as compatible solutes, they are essential for maintaining cellular turgor (by securing rigid structure of the cell wall) and they can scavenge free radicals. Storage polysaccharides (of which the main forms in algae and cyanobacteria are starch and glycogen) that serve as both energy and carbon source, buffer the disproportion between the carbohydrates production and consumption rates, and allow for active metabolism (e.g., nitrogen fixation) in the dark periods.

Carbohydrates are routinely analyzed in many life science laboratories. The saccharides content can be quantified by a wide range of chemical (e.g., chromatographic), biochemical (e.g. gravimetric, colorimetric, enzymatic) or physical (e.g., polarimetry) methods. The estimation of storage carbohydrates as described in this protocol represents a combination of starch/glycogen purification according to the previous studies (Schneegurt et al., 1994; Bandyopadhyay et al., 2010; Sinetova et al., 2012), starch/glycogen decomposition in acidic environment and free glucose determination by the classical phenol-sulfuric acid method (Dubois et al., 1956; Masuko et al., 2005). Total carbohydrates estimation consists of simple resuspension of the cellular pellet (after extraction of chlorophyll and carotenoids) in phenol solution and hydrolyzation of cellular saccharides by sulfuric acid.

The advantages of this protocol are simplicity, sensitivity and quickness. The carbohydrates/glycogen/starch estimation requires only several standard reagents, which makes this protocol much simpler and cheaper when compared to protocols that include enzymatic cleavage of glycogen (De Porcellinis et al., 2017; Khan et al., 2018), enzymatic determination of free glucose (Bandyopadhyay et al., 2010; Sinetova et al., 2012; Khan et al., 2018) or extensive amounts of chemicals (Khan et al., 2018). Another advantage of this protocol is that only 1 ml of diluted culture suspension is needed for the analysis (for further details see Note 1) which is significantly lower amount than required in other protocols (De Porcellinis et al., 2017). Additionally, saccharides/glycogen/starch measurement can be performed on the same samples that were originally used for determination of chlorophyll and carotenoids content (Sinetova et al., 2012).

On the other hand, this protocol is not as specific for determination of storage polysaccharides as the enzymatic assays (De Porcellinis et al., 2017) since glycogen or starch are distinguished only partially from other cellular polysaccharides (e.g., from polysaccharides of the cell wall). Another limitation is using D-glucose as a calibration standard since various carbohydrates that are present in the cells differ in the absorption spectra (Dubois et al., 1956; Masuko et al., 2005). Nevertheless, even with these limitations, this cheap, simple and fast protocol is suitable for rough estimation of carbohydrates content in algae and cyanobacteria.

Materials and Reagents

  1. Safe-lock tubes 1.5 ml SafeSeal (SARSTEDT, catalog number: 72.706.400 )
  2. Rotilabo® Sealing clips for the safe-lock tubes tubes (Carl Roth, catalog number: N217.1 )
  3. Holders for the safe-lock tubes (VWR, catalog number: 30128-282 )
  4. 96-well microplates (type P) with the original lids (Cole-Parmer, catalog numbers: EW-07903-80 and EW-07903-86 )
  5. Pipette tips
    1. Standard tips: 20-200 μl, 100-1,000 μl (Mettler-Toledo International, catalog numbers: 17001118 and 17001129 )
    2. Tips with filters: 20-200 μl (Mettler-Toledo International, catalog number: 17014963 )
  6. Reservoir for a multichannel pipette 60 ml (BrandTech Scientific, catalog number: 703459 )
  7. Aluminum foil (optional)
  8. Cyanobacterial/algae culture
  9. Methanol ≥ 99.9% (Alfa Aesar, catalog number: 41467.K7 )
  10. Phenol (Sigma-Aldrich, catalog number: P1037 )
  11. Potassium hydroxide p.a. (Ing. Petr Švec - PENTA, catalog number: 15520-31000 )
  12. Ethanol 96% (Merck, catalog number: 1590102500 )
  13. Sulfuric acid 96% (Ing. Petr Švec - PENTA, catalog number: 20370-11000 )
  14. D-glucose (Sigma-Aldrich, catalog number: G8270 )
  15. Distilled/double deionized water
  16. Sodium hydroxide (Ing. Petr Švec - PENTA, catalog number: 15760-31000 )
  17. Hydrochloric acid (Ing. Petr Švec - PENTA, catalog number: 19360-11000 )
  18. Glucose calibration series (see Recipes)

Equipment

  1. Pipettes
    1. 20-200 μl (Mettler-Toledo International, catalog number: 17014391 )
    2. 100-1,000 μl (Mettler-Toledo International, catalog number: 17014382 )
    3. Multichannel pipette 20-200 μl (Mettler-Toledo International, catalog number: 17013805 )
  2. Fridge 4 °C, freezer -20 °C (LIEBHERR, model: LCexv 4010 , catalog number: 9005382197172), optionally -80 °C (RevcoTM ExF -86 °C Upright Ultra-Low Temperature Freezer, Thermo Fisher Scientific, catalog number: EXF24086V )
  3. Fume hood (MERCI, model: M 1500 , catalog number: 2D100110200001)
  4. Refrigerated centrifuge (Sigma Laborzentrifugen, model: Sigma 1-16K , catalog number: 10030)
  5. Vacuum Concentrator (Eppendorf, model: Concentrator plus , catalog number: 5305000100)
  6. Analytical balances with an accuracy of 10 μg (Sartorius, catalog number: SECURA225D-1OBR )
  7. Microplate spectrophotometer (Thermo Fisher Scientific, model: MultiskanTM GO , catalog number: 51119300)

Procedure

  1. Harvesting biomass
    1. Harvest 1 ml of cyanobacterial culture suspension of OD730 0.1-0.5 (Note 1).
    2. Centrifuge the cells at 15,000 x g, laboratory temperature for 5 min and thoroughly discard the supernatant (Figure 1.1).
    3. For long-term storage, keep the samples at -80 °C and thaw on ice before further analysis.

  1. Extracting chlorophyll and carotenoids from the cells (Note 2)
    1. Add 1 ml of methanol, precooled to 4 °C. 
    2. Homogenize the sample by gentle pipetting up and down.
    3. In case of pigments quantification, cover the samples with aluminum foil. 
    4. Extract pigments from the cells by allowing the samples to sit at 4 °C in the dark for 20 min.
    5. Centrifuge the samples at 15,000 x g, 4 °C for 5 min and visually check pellet; it should be bluish (or purple) with no green color. If the pellet is green, repeat Steps B3-B4.
    6. Thoroughly discard all methanol supernatant by pipette (Figure 1.2). Keep the pellets for further analysis of total saccharides or starch/glycogen.
    7. Optionally, measure the concentration of chlorophyll and carotenoids in the methanol supernatant using a spectrophotometer according to Zavřel et al. (2015a). 
    8. For long-term storage, keep the pellets without chlorophyll and carotenoids at -80 °C and thaw on ice before further analysis.


      Figure 1. Individual steps of total carbohydrates and glycogen determination in Synechocystis sp. PCC 6803. The protocol begins with cells harvesting and centrifugation (1) and extraction of chlorophyll a and carotenoids to methanol (2). For total saccharides determination, the pellet is resuspended in 500 μl of distilled water and 500 μl of 5% phenol (3). For glycogen determination, the pellet is resuspended in 400 μl of 30% KOH (4), and the samples are incubated for 60 min at 95 °C to remove free glucose. After incubation, 1.2 ml of precooled ethanol is added (5) for overnight glycogen precipitation at -20 °C. After precipitation, samples are intensively centrifuged for 60 min, ethanol is discarded, pellet (6) is dried for 60 min under vacuum at 60 °C (7), and glycogen is hydrolyzed to glucose in 100 μl of 1 N HCl (8) at 95 °C. After hydrolysis, the samples are neutralized by addition of 100 μl of 1 N NaOH (9), diluted with 300 μl of distilled water (10) and mixed with 500 μl of 5% phenol (11). For quantification of free glucose, 8 x 60 μl of each sample is transferred to 96-well plate according to scheme in Figure 2 (12), and 150 μl of 96% sulfuric acid is added to each well for chromophores establishing (13). Cellular pellets after intensive centrifugation in ethanol (6) and drying (7) are marked by red arrows. The blue color of sample in step (3), originating in phycobiliproteins, does not influence the spectrophotometric quantification of total carbohydrates content.

  1. Preparing samples for determination of total cellular saccharides
    1. Sample the culture and extract chlorophyll and carotenoids from the cells as described in Procedure A and B.
    2. Add 500 μl of distilled water to each safe-lock tube with cellular pellet and mix well with a pipette.
    3. Move to the fume hood.
    4. Prepare 5% phenol (w/w) by dissolving 1 g of phenol in 19 ml of distilled water.
    5. Add 500 μl of 5% phenol to each safe-lock tube with cellular pellet and 500 μl of distilled water and mix well with a pipette (Figure 1.3).
    6. Keep the samples at laboratory temperature for 15 min (phenol will penetrate to the cells).
    7. Transfer 8 x 60 μl of each sample to the 96-well plate with a pipette according to the scheme in Figure 2 (positions A4-H12, each sample will have 8 technical replicates, Figure 1.12 and 1.13). In case of evaluating more than 9 samples, use an additional 96-well plate (Note 3).
    8. Continue with Procedure E.


      Figure 2. Schematic organization of the 96-well plate for measurement of total saccharides and starch/glycogen content. The first 24 wells (A1-H3) are designated for 6 points of glucose calibration (each calibration point will have 4 technical replicates), the remaining 72 wells (A4-H12) are designated for 9 samples (each sample will have 8 technical replicates).

  1. Preparing samples for starch/glycogen content determination
    1. Sample the culture and extract chlorophyll and carotenoids from the cells as described in the Procedure A and B.
    2. Prepare 30% KOH (w/w) by diluting 6 g of potassium hydroxide in 14 ml of distilled water.
    3. After thawing the cellular pellets on ice, add 400 μl of 30% KOH to each safe lock tube with cellular pellets and mix well with a pipette.
    4. Seal the safe-lock tubes with the sealing clips (Note 4).
    5. Incubate the samples at 95 °C for 90 min to remove free glucose and other alkali-sensitive saccharides (Evans, 1942; Whistler and BeMiller, 1958) (Figure 1.4).
    6. Cool down the samples at laboratory temperature for 10 min.
    7. Add 1.2 ml of ethanol precooled to 4 °C and mix well with a pipette (Figure 1.5).
    8. Store the samples overnight at -20 °C (Note 5).
    9. Centrifuge the samples at 4 °C, 20,000 x g for 60 min.
    10. Discard ethanol and keep the pellet for further analysis (Note 6, Figure 1.6).
    11. Dry the remaining ethanol under vacuum at 60 °C for 60 min (Figure 1.7).
    12. Add 100 μl of 1 N HCl to the dry pellet and mix well with a pipette (Figure 1.8).
    13. Seal the safe-lock tubes with the sealing clips (Note 4).
    14. Incubate the samples at 95 °C for 30 min to hydrolyze starch/glycogen.
    15. Cool down the samples at laboratory temperature for 10 min.
    16. Add 100 μl of 1 N NaOH and mix well with a pipette to neutralize 1 N HCl (Figure 1.9).
    17. Add 300 μl of distilled water and mix well with a pipette (Figure 1.10).
    18. Centrifuge at 15,000 x g at laboratory temperature for 10 min and transfer 500 μl of supernatant to a new safe-lock tube. Discard the safe-lock tube with pellet.
    19. Move to the fume hood.
    20. Add 500 μl of 5% phenol to each safe-lock tube with supernatant and mix well with a pipette (Figure 1.11).
    21. Keep the samples at laboratory temperature for 15 min (phenol will react with glucose).
    22. Transfer 8 x 60 μl of each sample to the 96-well plate with a pipette according to the scheme in Figure 1 (positions A4-H12, each sample will have 8 technical replicates, Figure 1.12 and 1.13). In case of evaluating more than 9 samples, use additional 96-well plate (Note 3).

  1. Preparing glucose calibration curve
    1. Prepare 6 x 500 μl of glucose calibration solutions (D-glucose: 25-500 μg ml-1) according to Table 1.

      Table 1. Preparation of calibration series of D-glucose in distilled water


    2. Move to the fume hood.
    3. Add 500 μl of 5% phenol solution to each tube with calibration solution and mix well with a pipette.
    4. Keep the tubes at laboratory temperature for 15 min (phenol will react with glucose)
    5. Transfer 4 x 60 μl of each glucose calibration solution to the 96-well plate with a pipette according to the scheme in Figure 1 (positions A1-H3, each calibration point will have 4 technical replicates) (Note 3, Figure 1.12 and 1.13).

  1. Spectrophotometric determination of saccharides content
    1. Pour 20 ml of 96% sulfuric acid to the reservoir for a multichannel pipette.
    2. Add 150 μl of 96% sulfuric acid to each well with the calibration solution or the sample using a multichannel pipette and mix well by pipetting up and down several times (Note 7).
    3. Cover the 96-well plate with the plastic lid.
    4. Incubate the samples at laboratory temperature for 5 min (Note 8).
    5. Measure the carbohydrates concentration by a microplate spectrophotometer.
    6. Recalculate the concentration of carbohydrates based on D-glucose calibration (Figure 3).


      Figure 3. Representative calibration of D-glucose (dissolved in distilled water) mixed with 5% phenol and 96% sulfuric acid, measured at 490 nm according to this protocol. Each calibration point represents the average of 4 technical replicates, the error bars represent standard deviations. The dotted line represents linear fit of the measured points calculated by the least squares method. The linear regression equation (y = αx + β) represents calibration equation slope (α) and intercept (β). The R2 coefficient was calculated by the least squares method.

Data analysis

The protocol provides 8 technical replicates of each sample and 4 technical replicates of each calibration solution. For reproducibility, it is strongly recommended to measure each sample at least in biological triplicates that should originate from independent experiments.
To determine the total carbohydrates and/or starch/glycogen concentration in the samples, the final absorbance at 490 nm (A490) should fit to the linear absorbance range of the spectrophotometer (Note 10). For the microplate spectrophotometer (MultiskanTM GO, Thermo Scientific), this linear absorbance range is 0.1-3.0. The final A490 of each sample should also fit to the range of the calibration curve. For glucose concentration 25-500 μg ml-1 as represented in Figure 3, this A490 range is 0.2-2.3.


Carbohydrates determination:
Calculate slope (α) and intercept (β) coefficients of the linear regression model (y = αx + β) from the calibration data (representative calibration curve is shown in Figure 3) and use these coefficients to calculate carbohydrates concentration in every sample and replicate according to equation (1) (Note 11):

In case of using lower or higher volumes of algae/cyanobacteria suspension than 1 ml (Step A1), and/or in case of diluting the samples by lower or higher volumes of distilled water than 500 μl (Step C2) or 300 μl (Step D17), and/or in case of adding lower or higher volumes of 5% phenol than 500 μl (Steps C5 and D20), calculate the final carbohydrates concentration according to the equation (2):

Representative results of glycogen content in Synechocystis sp. PCC 6803 that was cultivated under wide range of light intensities and sampled in exponential growth phase is shown in Figure 4.


Figure 4. Determination of glycogen content in cyanobacterium Synechocystis sp. PCC 6803. A. Representative measurement (raw data) of A490 within single 96-well plate, following scheme from Figure 2: A1-H3: six points of glucose calibration (25-500 μg ml-1), A4-H11: eight independent samples of Synechocystis cells that were cultivated under 27.5-1,100 μmolphotons m-2 sec-1 of red light. B. Final glycogen content in Synechocystis cells: the open circles represent averages from five independent experiments, the error bars represent standard deviations. Synechocystis was cultivated in a flat-panel photobioreactor (Nedbal et al., 2008) in a quasi-continuous cultivation regime under growth saturated conditions as described previously (Zavřel et al., 2015b). Glycogen was measured within five independent 96-well plates. The wells filled only with water (blanks, not used in this particular case) have A490 around 0.06.

Notes

  1. The amount of cell suspension required for analysis can vary with the culture density. Sample volume of 1 ml is optimized for cultures of Synechocystis sp. PCC 6803 with cell density approximately 1-2 x 107 cells ml-1. Such dense cultures of Synechocystis sp. PCC 6803 contain approximately 5-100 mg of both glycogen and total saccharides per liter culture. With diluted cultures, it is recommended to harvest bigger culture volume. On the contrary, for dense cultures lower sample volume is recommended–with high cellular densities, some pigments can remain in the cells after the methanol extraction, which can affect spectrophotometric quantification of total saccharides or starch/glycogen.
  2. This step should be omitted for eukaryotic algae, since pigment extraction by methanol without mechanic disruption of the robust cell walls is usually ineffective. For eukaryotic algae, pigments can be removed by overnight incubation of cellular pellets in ethanol, as described in Steps D7-D10.
  3. In case of analyzing more than 9 samples, perform the calibration for each single 96-well plate to secure identical conditions for both calibration solutions and samples.
  4. Sealing the safe-lock tubes with the sealing clips is critical since without this treatment the tubes will open during incubation at 95 °C and the samples will burst out.
  5. The precipitated starch/glycogen can be stored in ethanol at -20 °C for up to several months.
  6. The color of the precipitated starch/glycogen pellet can be ranging between yellow/brown and transparent. If the pellet is transparent, it is recommended to keep 100-200 μl of ethanol in the safe-lock tubes in order to avoid discarding of precipitated starch/glycogen together with ethanol (Step D10).
  7. Use pipette tips with filters to protect the pipette from the sulfuric acid aerosols. The reaction of 96% sulfuric acid with the samples releases significant amount of heat. It is therefore critical to pipette slowly to avoid spurting out of the final mixture from the wells. It is recommended to mix the phenol solution with sulfuric acid using the multichannel pipette for at least 8 times to secure reproducible conditions for all technical replicates and for all samples.
  8. The samples can be incubated for up to 40 min without significant loss of the final mixture color (Figure 5).


    Figure 5. Kinetics of reaction of D-glucose (dissolved in distilled water), 5% phenol and 96% sulfuric acid color in time, as measured by decrease of A490 signal. The dashed line represents linear fit of the measured points, calculated by the least squares method. The A490 slope (α) was calculated from the linear regression equation: y = αx.

  9. Absorption maximum of mixture of D-glucose (dissolved in distilled water), 5% phenol and 96% sulfuric acid is at 490 nm (Figure 6).


    Figure 6. Spectrum of mixture of D-glucose (dissolved in distilled water), 5% phenol and 96% sulfuric acid as prepared according to this protocol. The peak maximum is at 490 nm.

  10. In case A490 (the final carbohydrates concentration) is too high to fit to the linear absorbance range of the spectrophotometer, it is necessary to dilute the samples with 5% phenol (and equal amount of distilled water) before the chromophores establishing, i.e., before Step F2. Diluting the samples after chromophores establishing (after Step F2) can lead to incorrect spectrophotometric quantification of saccharides content.
  11. In case of starch concentration evaluation, divide the final value by factor 0.9, since the ratio of molar mass of starch (C6H10O5) and glucose (C6H12O6) is 0.9. For the representative calibration curve as shown in Figure 3, the carbohydrates concentration in the samples will be calculated as: carbohydrates [μg ml-1] = (A490-0.0727)/0.0045.

Recipes

  1. Glucose calibration series
    1. Prepare 500 μg ml-1 D-glucose (w/w) by dissolving 5 mg of D-glucose in 9.995 ml of distilled water
    2. Dilute the 500 μg ml-1 D-glucose solution by distilled water into 6 safe-lock tubes according to Table 1

Acknowledgments

The protocol was adopted from publication: Phenotypic characterization of Synechocystis sp. PCC 6803 substrains reveals differences in sensitivity to abiotic stress (Zavřel et al., 2017). T. Z., J. Č. and P. O. were supported by the Ministry of Education, Youth and Sports of the Czech Republic within the National Sustainability Program I (NPU I), grant number LO1415. J. Č. was also supported by GA ČR, Grant number 15-17367S. Access to instruments and other facilities was supported by the Czech research infrastructure for systems biology C4SYS (project No. LM2015055). M. A. S. was supported by a grant from the Russian Science Foundation (No. 14-14-00904). The authors declare no conflicts of interest.

References

  1. Bandyopadhyay, A., Stockel, J., Min, H., Sherman, L. A. and Pakrasi, H. B. (2010). High rates of photobiological H2 production by a cyanobacterium under aerobic conditions. Nat Commun 1: 139.
  2. De Porcellinis, A., Frigaard, N. U. and Sakuragi, Y. (2017). Determination of the glycogen content in cyanobacteria. J Vis Exp (125).
  3. Dubois, M., Gilles, K. A., Ton, J. K. H., Rebers, P. A. and Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Anal Chem 28: 350-356.
  4. Evans, W. L. (1942). Some less familiar aspects of carbohydrate chemistry. Chem Rev 31: 537-560.
  5. Khan, M. R., Wang, Y., Afrin, S., He, L. and Ma, G. (2018). Glycogen and extracellular glucose estimation from cyanobacteria Synechocystis sp. PCC 6803. Bio-Protocol 8(9): e2826.
  6. Masuko, T., Minami, A., Iwasaki, N., Majima, T., Nishimura, S. and Lee, Y. C. (2005). Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem 339(1): 69-72.
  7. Nedbal, L., Trtílek, M., Červený, J., Komárek, O. and Pakrasi, H. B. (2008). A photobioreactor system for precision cultivation of photoautotrophic microorganisms and for high-content analysis of suspension dynamics. Biotechnol Bioeng 100(5): 902-10.
  8. Raven, J. A. and Beardall, J. (2003). Carbohydrate metabolism and respiration in algae. In: Larkum, A. W. D., Douglas, S. E. and Raven, J. A. (Eds). Photosynthesis in Algae, Advances in Photosynthesis and Respiration. Springer Netherlands, Dordrecht, pp: 205-224.
  9. Schneegurt, M. A., Sherman, D. M., Nayar, S. and Sherman, L. A. (1994). Oscillating behavior of carbohydrate granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC 51142. J Bacteriol 176(6): 1586-1597.
  10. Sinetova, M. A., Červený, J., Zavřel, T. and Nedbal, L. (2012). On the dynamics and constraints of batch culture growth of the cyanobacterium Cyanothece sp. ATCC 51142. J Biotechnol 162(1): 148-155.
  11. Whistler, R. L. and Bemiller, J. N. (1958). Alkaline degradation of polysaccharides. Adv Carbohydr Chem 13: 289-329. 
  12. Zavřel, T., Očenášová, P. and Červený, J. (2017). Phenotypic characterization of Synechocystis sp. PCC 6803 substrains reveals differences in sensitivity to abiotic stress. PLoS One 12(12): e0189130.
  13. Zavřel, T., Sinetova, M. A., Búzová, D., Literáková, P. and Červený, J. (2015b). Characterization of a model cyanobacterium Synechocystis sp: PCC 6803 autotrophic growth in a flat-panel photobioreactor. Eng Life Sci 15(1): 122-132.
  14. Zavřel, T., Sinetova, M. A. and Červený, J. (2015a). Measurement of chlorophyll a and carotenoids concentration in cyanobacteria. Bio-protocol 5(9): e1467.

简介

这是用于定量测定藻类和蓝细菌中的储存和总碳水化合物的方案。该协议简单,快速,灵敏,只需要很少的标准化学品。该方案的最大优点是可以在已经用于叶绿素和类胡萝卜素定量的细胞沉淀中测定储存和总糖。由于建议一式三份进行颜料测量,因此每种颜料分析都可以生成总糖和糖原/淀粉含量定量的样品。

该方案用于量化蓝细菌 Synechocystis sp中的储存和总碳水化合物。 PCC 6803, Cyanothece sp。 ATCC 51142和 Cyanobacterium sp。 IPPAS B-1200。它还被用于估算 Galdieria (IPPAS P-500,IPPAS P-507,IPPAS P-508,IPPAS P-513), Cyanidium caldarium IPPAS中的储存多糖P-510,绿藻小球藻 sp。 IPPAS C-1和C-1210, Parachlorella kessleri IPPAS C-9, Nannochloris sp。 C-1509, Coelastrella sp。 IPPAS H-626, Haematococcus sp。 IPPAS H-629和H-239,以及 Eustigmatos sp。 IPPAS H-242和IPPAS C-70。

【背景】碳水化合物在藻类和蓝细菌的代谢中起着许多作用。正如Raven和Beardall(Raven和Beardall,2003)所总结的那样,碳水化合物代表碳还原/氧化途径(光合作用和光呼吸)中间体的主要汇集,它们提供生长所需的骨架(例如,for氨基酸或细胞壁生物合成),它们代表ATP和还原当量的来源(通过呼吸途径),它们作为相容的溶质,它们对维持细胞膨胀是必不可少的(通过确保细胞壁的刚性结构)并且它们可以清除自由基。储存多糖(其中藻类和蓝藻中的主要形式是淀粉和糖原)作为能量和碳源,缓冲碳水化合物生产和消费率之间的不成比例,并允许活跃的新陈代谢(例如,固氮)在黑暗时期。

在许多生命科学实验室中常规分析碳水化合物。糖类含量可以通过各种化学品(例如,色谱法),生化(例如重量法,比色法,酶法)或物理(例如,polarimetry)方法。本方案中描述的储存碳水化合物的估计代表了根据先前研究的淀粉/糖原纯化的组合(Schneegurt 等人,1994; Bandyopadhyay 等人, 2010; Sinetova et al。,2012),酸性环境中淀粉/糖原分解和经典苯酚 - 硫酸法测定游离葡萄糖(Dubois et al。,1956) ; Masuko et al。,2005)。总碳水化合物估计包括在苯酚溶液中简单地再悬浮细胞沉淀(在提取叶绿素和类胡萝卜素之后)和通过硫酸水解细胞糖。

该协议的优点是简单,灵敏和快速。碳水化合物/糖原/淀粉估计仅需要几种标准试剂,与包括酶促切割糖原的方案相比,这使得该方案更简单,更便宜(De Porcellinis et al。,2017; Khan et al。,2018),酶法测定游离葡萄糖(Bandyopadhyay et al。,2010; Sinetova et al。,2012; Khan et al。,2018)或大量化学品(Khan et al。,2018)。该方案的另一个优点是分析只需要1 ml稀释的培养悬浮液(详见附注1),其数量明显低于其他方案所需的量(De Porcellinis et al。 ,2017)。另外,糖类/糖原/淀粉测量可以在最初用于测定叶绿素和类胡萝卜素含量的相同样品上进行(Sinetova 等人,,2012)。

另一方面,该方案不像酶测定法那样特异于测定储存多糖(De Porcellinis et al。,2017),因为糖原或淀粉仅部分区别于其他细胞多糖(< em>例如,来自细胞壁的多糖)。另一个限制是使用D-葡萄糖作为校准标准,因为细胞中存在的各种碳水化合物的吸收光谱不同(Dubois et al。,1956; Masuko et al。,2005)。然而,即使有这些限制,这种便宜,简单和快速的方案也适用于粗略估计藻类和蓝细菌中的碳水化合物含量。

关键字:糖, 碳水化合物, 多糖, 比色法, 分光光度法, 蓝藻, 小球藻, 红球藻

材料和试剂

  1. 安全锁管1.5 ml SafeSeal(SARSTEDT,目录号:72.706.400)
  2. Rotilabo ®安全锁管用密封夹(Carl Roth,目录号:N217.1)
  3. 安全锁管支架(VWR,目录号:30128-282)
  4. 带有原始盖子的96孔微孔板(P型)(Cole-Parmer,目录号:EW-07903-80和EW-07903-86)
  5. 移液器吸头
    1. 标准提示:20-200μl,100-1,000μl(Mettler-Toledo International,目录号:17001118和17001129)
    2. 过滤器提示:20-200μl(Mettler-Toledo International,目录号:17014963)
  6. 用于多通道移液器的储液器60 ml(BrandTech Scientific,目录号:703459)
  7. 铝箔(可选)
  8. 蓝藻/藻类培养
  9. 甲醇≥99.9%(Alfa Aesar,目录号:41467.K7)
  10. 苯酚(Sigma-Aldrich,目录号:P1037)
  11. 氢氧化钾 p.a。(Ing.PetrŠvec - PENTA,目录号:15520-31000)
  12. 乙醇96%(默克,目录号:1590102500)
  13. 硫酸96%(Ing.PetrŠvec - PENTA,目录号:20370-11000)
  14. D-葡萄糖(Sigma-Aldrich,目录号:G8270)
  15. 蒸馏/双去离子水
  16. 氢氧化钠(Ing.PetrŠvec - PENTA,目录号:15760-31000)
  17. 盐酸(Ing.PetrŠvec - PENTA,目录号:19360-11000)
  18. 葡萄糖校准系列(见食谱)

设备

  1. 移液器
    1. 20-200μl(Mettler-Toledo International,目录号:17014391)
    2. 100-1,000μl(Mettler-Toledo International,目录号:17014382)
    3. 多通道移液器20-200μl(Mettler-Toledo International,目录号:17013805)
  2. 冰箱4°C,冷冻室-20°C(LIEBHERR,型号:LCexv 4010,目录号:9005382197172),可选-80°C(Revco TM ExF -86°C立式超低温冰箱,Thermo Fisher Scientific,目录号:EXF24086V)
  3. 通风橱(MERCI,型号:M 1500,目录号:2D100110200001)
  4. 冷冻离心机(Sigma Laborzentrifugen,型号:Sigma 1-16K,目录号:10030)
  5. 真空浓缩器(Eppendorf,型号:Concentrator plus,目录号:5305000100)
  6. 分析天平,精确度为10μg(赛多利斯,目录号:SECURA225D-1OBR)
  7. 微孔板分光光度计(Thermo Fisher Scientific,型号:Multiskan TM GO,目录号:51119300)

程序

  1. 收获生物量
    1. 收获1ml OD 730 0.1-0.5的蓝藻培养物悬浮液(注1)。
    2. 将细胞以15,000 x g 离心,实验室温度5分钟,彻底弃去上清液(图1.1)。
    3. 对于长期储存,在进一步分析之前,将样品保持在-80°C并在冰上融化。

  1. 从细胞中提取叶绿素和类胡萝卜素(注2)
    1. 加入1毫升甲醇,预冷至4°C&nbsp;
    2. 通过轻轻向上和向下移液使样品均匀化。
    3. 在颜料定量的情况下,用铝箔覆盖样品。&nbsp;
    4. 通过使样品在4℃在黑暗中静置20分钟从细胞中提取颜料。
    5. 将样品在15,000 x g ,4°C下离心5分钟,目测检查颗粒;它应该是蓝色(或紫色),没有绿色。如果颗粒为绿色,则重复步骤B3-B4。
    6. 用移液管彻底弃去所有甲醇上清液(图1.2)。保留颗粒以进一步分析总糖或淀粉/糖原。
    7. 任选地,根据Zavřel等(2015a),使用分光光度计测量甲醇上清液中叶绿素和类胡萝卜素的浓度。&nbsp;
    8. 对于长期储存,在-80°C下保持没有叶绿素和类胡萝卜素的颗粒,并在进一步分析前在冰上融化。


      图1. Synechocystis sp。中总碳水化合物和糖原测定的各个步骤PCC 6803。该方案从细胞收获和离心(1)开始,并将叶绿素a和类胡萝卜素提取到甲醇(2)。对于总糖测定,将沉淀重悬于500μl蒸馏水和500μl5%苯酚(3)中。对于糖原测定,将沉淀重悬于400μl30%KOH(4)中,并将样品在95℃温育60分钟以除去游离葡萄糖。温育后,加入1.2ml预冷的乙醇(5),在-20℃下进行过夜的糖原沉淀。沉淀后,将样品强烈离心60分钟,弃去乙醇,将沉淀(6)在60℃下真空干燥60分钟(7),并将糖原在100μl1NHCl中水解成葡萄糖(8)在95°C。水解后,通过加入100μl1NNaOH(9)中和样品,用300μl蒸馏水(10)稀释并与500μl5%苯酚(11)混合。为了定量游离葡萄糖,根据图2(12)中的方案将8×60μl的每种样品转移到96孔板中,并向每个孔中加入150μl96%硫酸用于生色团建立(13)。用乙醇(6)和干燥(7)强力离心后的细胞沉淀用红色箭头标记。步骤(3)中的样品的蓝色,源自藻胆蛋白,不影响总碳水化合物含量的分光光度定量。

  1. 准备样品以测定总细胞糖类
    1. 如过程A和B中所述,对细胞进行取样并从细胞中提取叶绿素和类胡萝卜素。
    2. 向每个带有细胞沉淀的安全锁管中加入500μl蒸馏水,并用移液管充分混合。
    3. 移动到通风橱。
    4. 通过将1g苯酚溶解在19ml蒸馏水中制备5%苯酚(w / w)。
    5. 将500μl5%苯酚加入每个带有细胞沉淀和500μl蒸馏水的安全锁管中,并用移液管充分混合(图1.3)。
    6. 将样品保持在实验室温度15分钟(苯酚将渗透到细胞中)。
    7. 根据图2中的方案用移液管将8×60μl的每个样品转移到96孔板中(位置A4-H12,每个样品将具有8个技术重复,图1.12和1.13)。如果评估超过9个样品,请使用额外的96孔板(注3)。
    8. 继续执行程序E.


      图2.用于测量总糖和淀粉/糖原含量的96孔板的示意组织。前24个孔(A1-H3)指定用于6个葡萄糖校准点(每个校准点)将有4个技术重复),其余72个孔(A4-H12)被指定用于9个样品(每个样品将有8个技术重复)。

  1. 准备淀粉/糖原含量测定样品
    1. 如过程A和B中所述,对培养物进行取样并从细胞中提取叶绿素和类胡萝卜素。
    2. 通过在14ml蒸馏水中稀释6g氢氧化钾制备30%KOH(w / w)。
    3. 在冰上解冻细胞沉淀后,向每个带有细胞沉淀的安全锁管中加入400μl30%KOH,并用移液管充分混合。
    4. 用密封夹密封安全锁管(注4)。
    5. 将样品在95°C孵育90分钟以除去游离葡萄糖和其他碱敏感糖(Evans,1942; Whistler和BeMiller,1958)(图1.4)。
    6. 在实验室温度下冷却样品10分钟。
    7. 加入预先冷却至4°C的1.2 ml乙醇,用移液管充分混合(图1.5)。
    8. 将样品在-20°C下保存过夜(注5)。
    9. 将样品在4℃,20,000 x g 下离心60分钟。
    10. 弃去乙醇并保留颗粒用于进一步分析(注6,图1.6)。
    11. 将剩余的乙醇在真空下在60℃下干燥60分钟(图1.7)。
    12. 向干燥的沉淀中加入100μl1NHCl,并用移液管充分混合(图1.8)。
    13. 用密封夹密封安全锁管(注4)。
    14. 将样品在95℃孵育30分钟以水解淀粉/糖原。
    15. 在实验室温度下冷却样品10分钟。
    16. 加入100μl1NNaOH并用移液管充分混合以中和1N HCl(图1.9)。
    17. 加入300μl蒸馏水,用移液管充分混合(图1.10)。
    18. 在实验室温度下以15,000 x g 离心10分钟,并将500μl上清液转移到新的安全锁定管中。丢弃带有颗粒的安全锁管。
    19. 移动到通风橱。
    20. 用上清液向每个安全锁管中加入500μl5%苯酚,并用移液管充分混合(图1.11)。
    21. 将样品保持在实验室温度15分钟(苯酚将与葡萄糖反应)。
    22. 根据图1中的方案用移液管将8×60μl的每种样品转移到96孔板中(位置A4-H12,每个样品将具有8个技术重复,图1.12和1.13)。如果评估超过9个样品,请使用额外的96孔板(注3)。

  1. 准备葡萄糖校准曲线
    1. 根据表1准备6 x500μl葡萄糖校准溶液(D-葡萄糖:25-500μg/ ml -1 )。

      表1.蒸馏水中D-葡萄糖校准系列的制备


    2. 移动到通风橱。
    3. 用校准溶液向每个管中加入500μl5%苯酚溶液,并用移液管充分混合。
    4. 将试管保持在实验室温度15分钟(苯酚会与葡萄糖反应)
    5. 根据图1中的方案,用移液管将4 x60μl的每种葡萄糖校准溶液转移至96孔板(位置A1-H3,每个校准点将有4个技术重复)(注3,图1.12和1.13) 。

  1. 分光光度法测定糖类含量
    1. 将20毫升96%的硫酸倒入容器中,用于多通道移液器。
    2. 用校准溶液或样品使用多通道移液管向每个孔中加入150μl96%硫酸,并通过上下移液几次混合均匀(注7)。
    3. 用塑料盖盖住96孔板。
    4. 在实验室温度下孵育样品5分钟(注8)。
    5. 用微孔板分光光度计测量碳水化合物浓度:
    6. 根据D-葡萄糖校准重新计算碳水化合物的浓度(图3)。


      图3.根据该方案在490 nm处测量的D-葡萄糖(溶解在蒸馏水中)与5%苯酚和96%硫酸混合的代表性校准。每个校准点代表4的平均值技术重复,误差线表示标准偏差。虚线表示通过最小二乘法计算的测量点的线性拟合。线性回归方程(y =αx+β)表示校准方程斜率(α)和截距(β)。通过最小二乘法计算R 2 系数。

数据分析

该协议提供每个样品的8个技术重复和每个校准溶液的4个技术重复。为了重现性,强烈建议至少在生物学一式三份中测量每个样品,这些样品应来自独立实验。
为了确定样品中的总碳水化合物和/或淀粉/糖原浓度,490nm处的最终吸光度(A <4> )应符合分光光度计的线性吸光度范围(注释10)。对于微孔板分光光度计(Multiskan TM GO,Thermo Scientific),该线性吸光度范围为0.1-3.0。每个样品的最终A 490 也应符合校准曲线的范围。对于葡萄糖浓度25-500μg/ ml,如图3所示,该A 490 范围是0.2-2.3。

碳水化合物测定:
从校准数据计算线性回归模型(y =αx+β)的斜率(α)和截距(β)系数(代表性校准曲线如图3所示),并使用这些系数计算每个样品中的碳水化合物浓度并复制根据等式(1)(注11):

如果使用比1 ml更低或更高体积的藻类/蓝细菌悬浮液(步骤A1),和/或使用比500μl(步骤C2)或300μl更低或更高体积的蒸馏水稀释样品的情况(步骤D17),和/或在添加5%或更高体积的5%苯酚而不是500μl的情况下(步骤C5和D20),根据等式(2)计算最终碳水化合物浓度:

Synechocystis sp。中糖原含量的代表性结果。在广泛的光照强度下培养并在指数生长期采样的PCC 6803如图4所示。

图4.蓝藻Synechocystis sp。中糖原含量的测定PCC 6803。 A.单个96孔板中A 490 的代表性测量(原始数据),遵循图2的方案:A1-H3:六个葡萄糖校准点(25- 500μg/ ml -1 ),A4-H11:8个独立的 Synechocystis 细胞样本,在27.5-1,100μmol光子 m -2 sec -1 。 B. Synechocystis 细胞中的最终糖原含量:空心圆表示来自五个独立实验的平均值,误差条代表标准偏差。 Synechocystis 在平板光生物反应器(Nedbal et al。,2008)中以准连续培养方式在如前所述的生长饱和条件下培养(Zavřel等人。,2015b)。在五个独立的96孔板内测量糖原。仅用水填充的孔(在这种特定情况下不使用的坯料)具有约<4> 的0.06左右。

笔记

  1. 分析所需的细胞悬浮液的量可随培养密度而变化。对于 Synechocystis sp的培养物优化1ml的样品体积。 PCC 6803,细胞密度约为1-2×10 7,细胞ml -1 。这种 Synechocystis sp的密集培养物。 PCC 6803每升培养物含有约5-100mg的糖原和总糖。使用稀释培养物,建议收获更大的培养体积。相反,对于致密培养物,建议使用较低的样品体积 - 具有高细胞密度,在甲醇提取后一些色素可以保留在细胞中,这可以影响总糖或淀粉/糖原的分光光度定量。
  2. 对于真核藻类,应该省略该步骤,因为通过甲醇进行色素提取而没有机械破坏坚固的细胞壁通常是无效的。对于真核藻类,可以通过在乙醇中过夜孵育细胞沉淀来除去色素,如步骤D7-D10中所述。
  3. 如果分析超过9个样品,请对每个96孔板进行校准,以确保校准溶液和样品的相同条件。
  4. 用密封夹密封安全锁管是至关重要的,因为如果没有这种处理,在95℃温育期间管将打开并且样品将爆裂。
  5. 沉淀的淀粉/糖原可以在-20℃下储存在乙醇中达数月。
  6. 沉淀淀粉/糖原颗粒的颜色可以在黄色/棕色和透明之间。如果颗粒是透明的,建议在安全锁管中保留100-200μl乙醇,以避免与乙醇一起丢弃沉淀的淀粉/糖原(步骤D10)。
  7. 使用带过滤器的移液器吸头保护移液器免受硫酸气溶胶的影响。 96%硫酸与样品的反应释放出大量的热量。因此,必须缓慢移液以避免从孔中喷出最终混合物。建议使用多通道移液管将苯酚溶液与硫酸混合至少8次,以确保所有技术重复和所有样品的可重复条件。
  8. 样品可以孵育长达40分钟而不会显着损失最终的混合物颜色(图5)。


    图5.通过A 490 信号的减少测量的D-葡萄糖(溶于蒸馏水),5%苯酚和96%硫酸颜色的反应动力学。虚线表示通过最小二乘法计算的测量点的线性拟合。 A 490 斜率(α)由线性回归方程计算:y =αx。

  9. D-葡萄糖(溶于蒸馏水),5%苯酚和96%硫酸的混合物的吸收最大值为490nm(图6)。


    图6.根据本方案制备的D-葡萄糖(溶于蒸馏水),5%苯酚和96%硫酸的混合物的光谱。峰值最大值为490 nm。
  10. 如果A 490 (最终碳水化合物浓度)太高而不符合分光光度计的线性吸光度范围,则必须用5%苯酚(和等量蒸馏水)稀释样品在生色团建立之前,即,在步骤F2之前。在生色团建立后(步骤F2之后)稀释样品可导致不正确的分光光度定量糖类含量。
  11. 在淀粉浓度评估的情况下,将最终值除以因子0.9,因为淀粉的摩尔质量比(C 6 H 10 O 5 )和葡萄糖(C 6 H 12 O 6 )为0.9。对于如图3所示的代表性校准曲线,样品中的碳水化合物浓度计算如下:碳水化合物[μgml -1 ] =(A 490 -0.0727) /0.0045。

食谱

  1. 葡萄糖校准系列
    1. 将5毫克D-葡萄糖溶于9.995毫升蒸馏水中,制备500微克 -1 D-葡萄糖(w / w)
    2. 根据表1,用蒸馏水将500μg/ ml -1 D-葡萄糖溶液稀释到6个安全锁管中

致谢

该方案采用自出版物: Synechocystis sp的表型特征。 PCC 6803亚基显示出对非生物胁迫的敏感性差异(Zavřel et al。,2017)。 T. Z.,J。J.在国家可持续发展计划I(NPU I)中,捷克共和国教育,青年和体育部支持P. O.,拨款号为LO1415。 J.Č。也得到了GAČR的支持,授权号为15-17367S。捷克的系统生物学C4SYS研究基础设施(项目编号LM2015055)支持使用仪器和其他设施。 M. A. S.得到了俄罗斯科学基金会(第14-14-00904号)的资助。作者宣称没有利益冲突。

参考

  1. Bandyopadhyay,A.,Stockel,J.,Min,H.,Sherman,L.A。和Pakrasi,H.B。(2010)。 在有氧条件下蓝藻产生的光生物H2产生率很高。 Nat传播 1:139。
  2. De Porcellinis,A.,Frigaard,N。U.和Sakuragi,Y。(2017)。 确定蓝细菌中的糖原含量。 J Vis Exp (125)。
  3. Dubois,M.,Gilles,K.A.,Ton,J。K.H.,Rebers,P。A. and Smith,F。(1956)。 用于测定糖和相关物质的比色法。 Anal Chem 28:350-356。
  4. 埃文斯,W。L.(1942)。 一些不太熟悉的碳水化合物化学方面。 Chem Rev 31:537-560。
  5. Khan,M.R.,Wang,Y.,Afrin,S.,He,L。和Ma,G。(2018)。 从蓝细菌 Synechocystis sp。估算糖原和细胞外葡萄糖。 PCC 6803. 生物协议 8(9):e2826。
  6. Masuko,T.,Minami,A.,Iwasaki,N.,Majima,T.,Nishimura,S。和Lee,Y.C。(2005)。 采用微孔板形式的苯酚 - 硫酸法进行碳水化合物分析。 肛门Biochem 339(1):69-72。
  7. Nedbal,L.,Trtílek,M.,Červený,J.,Komárek,O。和Pakrasi,H。B.(2008)。 光生物反应器系统,用于精确培养光合自养微生物和高悬浮动力学分析。 Biotechnol Bioeng 100(5):902-10。
  8. Raven,J。A.和Beardall,J。(2003)。 藻类中的碳水化合物代谢和呼吸。在:Larkum, AWD,Douglas,SE和Raven,JA(编辑)。藻类的光合作用,光合作用和呼吸作用的进展。 Springer Netherlands,Dordrecht,pp:205-224。
  9. Schneegurt,M。A.,Sherman,D。M.,Nayar,S。和Sherman,L。A.(1994)。 氰化青蟹Cyanothece sp。中碳水化合物颗粒形成和二氮固定的振荡行为。菌株ATCC 51142. J Bacteriol 176(6):1586-1597。
  10. Sinetova,M.A.,Červený,J.,Zavřel,T。和Nedbal,L。(2012)。 关于蓝藻 Cyanothece sp的分批培养生长的动态和限制。 ATCC 51142. J Biotechnol 162(1):148-155。
  11. Whistler,R。L.和Bemiller,J。N.(1958)。 多糖的碱性降解。 Adv Carbohydr Chem 13: 。289-329&NBSP;
  12. Zavřel,T.,Očenášová,P。和Červený,J。(2017)。 Synechocystis sp的表型特征。 PCC 6803亚基显示出对非生物胁迫的敏感性差异。 PLoS One 12(12):e0189130。
  13. Zavřel,T.,Sinetova,M。A.,Búzová,D.,Literáková,P。和Červený,J。(2015b)。 模型蓝藻 Synechocystis sp:PCC 6803自养生长的表征平板光生物反应器。 Eng Life Sci 15(1):122-132。
  14. Zavřel,T.,Sinetova,M。A.和Červený,J。(2015a)。 测量蓝藻中的叶绿素a和类胡萝卜素浓度。 生物协议 5(9 ):e1467。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zavřel, T., Očenášová, P., Sinetova, M. A. and Červený, J. (2018). Determination of Storage (Starch/Glycogen) and Total Saccharides Content in Algae and Cyanobacteria by a Phenol-Sulfuric Acid Method. Bio-protocol 8(15): e2966. DOI: 10.21769/BioProtoc.2966.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。