Difference between revisions of "Measurement of Glycogen in Microbiota"
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==== '''This method is adapted from: "'''Optimisation of glycogen quantification in mixed microbial cultures" 2012<ref>{{Cite journal|date=2012-08-01|title=Optimisation of glycogen quantification in mixed microbial cultures|url=https://www.sciencedirect.com/science/article/pii/S0960852412008401?via=ihub|journal=Bioresource Technology|language=en|volume=118|doi=10.1016/j.biortech.2012.05.087|issn=0960-8524}}</ref> ==== | ==== '''This method is adapted from: "'''Optimisation of glycogen quantification in mixed microbial cultures" 2012<ref>{{Cite journal|date=2012-08-01|title=Optimisation of glycogen quantification in mixed microbial cultures|url=https://www.sciencedirect.com/science/article/pii/S0960852412008401?via=ihub|journal=Bioresource Technology|language=en|volume=118|doi=10.1016/j.biortech.2012.05.087|issn=0960-8524}}</ref> ==== | ||
| − | ==== '''This method was used for:''' Quantifying Glycogen in Solids at Full-Scale Enhanced Biological Phosphorous Removal Wastewater Facilities, 2018<ref>{{Cite journal|last=RedCorn, Engelberth|first=|date=2018|title=Quantifying Glycogen in Solids at Full-Scale Enhanced Biological Phosphorous Removal Wastewater Facilities|url=https://ascelibrary.org/doi/abs/10.1061/%28ASCE%29EE.1943-7870.0001438|journal=Journal of Environmental Engineering|volume=144|pages=Issue 9|via=}}</ref> ==== | + | ==== '''This method was used for:''' Quantifying Glycogen in Solids at Full-Scale Enhanced Biological Phosphorous Removal Wastewater Facilities, 2018<ref name=":0">{{Cite journal|last=RedCorn, Engelberth|first=|date=2018|title=Quantifying Glycogen in Solids at Full-Scale Enhanced Biological Phosphorous Removal Wastewater Facilities|url=https://ascelibrary.org/doi/abs/10.1061/%28ASCE%29EE.1943-7870.0001438|journal=Journal of Environmental Engineering|volume=144|pages=Issue 9|via=}}</ref> ==== |
== Discussion ==<!-- Discuss the purpose, the suitability of the method for certain situations, and the fundamentals science behind the method.--> | == Discussion ==<!-- Discuss the purpose, the suitability of the method for certain situations, and the fundamentals science behind the method.--> | ||
| − | Samples | + | Samples are prepped by preserving with formaldehyde prior to lyophilizing; this is the same as methods measuring PHA and therefore the lyophilized sample can be used for both glycogen and PHA analysis downstream. If HPLC is used for the final measurement then the total glucose is assumed to all come from glycogen. If other carbohydrate analysis methods are used (e.g. Phenol-Sulfuric Acid method) then there is a greater potential of interference from non-glucose carbohydrates. |
== Materials and Equipment ==<!-- Any tubes, pipette tips ect. --> | == Materials and Equipment ==<!-- Any tubes, pipette tips ect. --> | ||
| Line 76: | Line 76: | ||
==Precision and Interference== | ==Precision and Interference== | ||
| − | <references /> | + | RedCorn & Engelberth indicated that a potential 5% increase in final glucose measured could be due to degradation of cellulose when measuring activated sludge samples<ref name=":0" /><references /> |
Revision as of 03:50, 1 March 2019
Contents
- 1 Authors:
- 2 This method is also called:
- 3 This method is adapted from: "Optimisation of glycogen quantification in mixed microbial cultures" 2012[1]
- 4 This method was used for: Quantifying Glycogen in Solids at Full-Scale Enhanced Biological Phosphorous Removal Wastewater Facilities, 2018[2]
- 5 Discussion
- 6 Materials and Equipment
- 7 Reagents
- 8 Procedure
- 9 Common Mistakes
- 10 Precision and Interference
Authors:
This method is also called:
This method is adapted from: "Optimisation of glycogen quantification in mixed microbial cultures" 2012[1]
This method was used for: Quantifying Glycogen in Solids at Full-Scale Enhanced Biological Phosphorous Removal Wastewater Facilities, 2018[2]
Discussion
Samples are prepped by preserving with formaldehyde prior to lyophilizing; this is the same as methods measuring PHA and therefore the lyophilized sample can be used for both glycogen and PHA analysis downstream. If HPLC is used for the final measurement then the total glucose is assumed to all come from glycogen. If other carbohydrate analysis methods are used (e.g. Phenol-Sulfuric Acid method) then there is a greater potential of interference from non-glucose carbohydrates.
Materials and Equipment
- Fume Hood
- Lyophilizer
- 50ml centrifuge tubes (e.g. Falcon Tubes)
Reagents
| Reagent | Concentration | Units | CAS Number |
|---|---|---|---|
| Formaldehyde | 37 | % | 50-00-0 |
| PBS Solution | |||
| HCl | 0.9 | Molar | 7647-01-0 |
Procedure
- Label Tubes;
- While in a fume hood, add 4 to 5 drops (0.25 ml) of 37% formaldehyde to the 50ml centrifuge tube. This will act as a sample preserving agent.
- Place aliquots of 40 ml per sample in 50 ml centrifuge tubes.
- Collect sludge in a 15 ml centrifuge tube (should be about 20 mg solids worth) through one of the following methods;
- Store at 4°C for 2 hours
- Wash the sample as follows to remove paraformaldehyde or formaldehyde; Centrifuge at 3000 RCF for 5 min, and carefully decant the supernatant. Add 10 ml of PBS solution and vortex sample to suspend pellet. Centrifuge at 3000 RCF for 5 min, and carefully decant the supernatant. Add 10 ml of PBS solution and vortex sample to suspend pellet. Centrifuge at 3000 RCF for 5 min, and carefully decant the supernatant. Centrifuge Sample at 5000 rcf for 5min, decant supernatant
- Cover with parafilm and poke holes in the parafilm. Place cap on over parafilm. Freeze sample in -80°C Freezer.
- Start the lyophlizer cooling unit and run for 10 min prior to running.
- Remove cap from sample. Place sample in lyophilizer and start the vacuum pump. Ensure vacuum is being supplied to lyophlilizer container, which contains the sample. Lyophlize overnight.
- When removing from lyophilizer, make sure the lyophilizer lid is off in order to let lyophilizer dry out.
- Place 45 mg of lyophilized matter into a 50ml centrifuge tube. Label the tube.
- Place 45ml of 0.9 M HCl (or load to 1mg/ml if less than 45 mg lyophilized biosolids)
- Place in oven for 3h at 100°C
- Cool Sample in an ice bath prior to HPLC analysis - HCl solution can be measured directly on HPLC via Measurement of Sugars and Alcohol on HPLC.
Common Mistakes
| Mistake | Evidence of the Mistake | Cause | Effect on Results |
|---|---|---|---|
| Leave valve to lyophilzer jar | Cooling coils on lyophilizer have excessive ice | Moisture from the air allowed to enter. | Cannot complete lyophilization |
Precision and Interference
RedCorn & Engelberth indicated that a potential 5% increase in final glucose measured could be due to degradation of cellulose when measuring activated sludge samples[2]
- ↑ "Optimisation of glycogen quantification in mixed microbial cultures". Bioresource Technology. 118. 2012-08-01. doi:10.1016/j.biortech.2012.05.087. ISSN 0960-8524.
- ↑ 2.0 2.1 RedCorn, Engelberth (2018). "Quantifying Glycogen in Solids at Full-Scale Enhanced Biological Phosphorous Removal Wastewater Facilities". Journal of Environmental Engineering. 144: Issue 9.
