Difference between revisions of "Measurement of Glycogen in Microbiota"
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==== '''This method is also called:''' ==== | ==== '''This method is also called:''' ==== | ||
| − | ==== '''This method is adapted from: < | + | ==== '''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> ==== | ||
== 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 can be prepped by preserving with formaldehyde prior to lyophilizing the same as methods measuring PHA. | ||
== Materials and Equipment ==<!-- Any tubes, pipette tips ect. --> | == Materials and Equipment ==<!-- Any tubes, pipette tips ect. --> | ||
| + | * Fume Hood | ||
| + | * Lyophilizer | ||
| + | * 50ml centrifuge tubes (e.g. Falcon Tubes) | ||
==Reagents==<!-- Test kits go here if used--> | ==Reagents==<!-- Test kits go here if used--> | ||
| Line 21: | Line 27: | ||
! CAS Number | ! CAS Number | ||
|- | |- | ||
| − | | <!-- Common Name--> | + | | Formaldehyde<!-- Common Name--> |
| − | | <!-- A value--> | + | | 37<!-- A value--> |
| − | | <!-- By weight or volume? g/L, Mol, ect..--> | + | | <!-- By weight or volume? g/L, Mol, ect..-->% |
| − | | <!-- Universal Identifier, regardless of manufacturer --> | + | | 50-00-0<!-- Universal Identifier, regardless of manufacturer --> |
| + | |- | ||
| + | |PBS Solution | ||
| + | | | ||
| + | | | ||
| + | | | ||
| + | |- | ||
| + | |HCl | ||
| + | |0.9 | ||
| + | |Molar | ||
| + | |7647-01-0 | ||
|} | |} | ||
==Procedure== | ==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 | ||
| + | # b. Add 10 ml of PBS solution and vortex sample to suspend pellet | ||
| + | # c. Centrifuge at 3000 RCF for 5 min, and carefully decant the supernatant | ||
| + | # d. Add 10 ml of PBS solution and vortex sample to suspend pellet | ||
| + | # e. Centrifuge at 3000 RCF for 5 min, and carefully decant the supernatant | ||
| + | # 8. Centrifuge Sample at 5000 rcf for 5min, decant supernatant | ||
| + | # 9. Cover with parafilm and poke holes in the parafilm. Place cap on over parafilm. Freeze sample in -80°C Freezer. | ||
| + | # 10. Start the lyophlizer cooling unit and run for 10 min prior to running. | ||
| + | # 11. 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. | ||
| + | # 12. When removing from lyophilizer, make sure the lyophilizer lid is off in order to let lyophilizer dry out. | ||
| + | # 13. Place 45 mg of lyophilized matter into a 50ml centrifuge tube. Label the tube. | ||
| + | # 14. Place 45ml of 0.9 M HCl (or load to 1mg/ml if less than 45 mg lyophilized biosolids) | ||
| + | # 15. Place in oven for 3h at 100°C | ||
| + | # 16. Cool Sample in an ice bath prior to HPLC analysis | ||
==Common Mistakes== | ==Common Mistakes== | ||
Revision as of 03:41, 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 can be prepped by preserving with formaldehyde prior to lyophilizing the same as methods measuring PHA.
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
- b. Add 10 ml of PBS solution and vortex sample to suspend pellet
- c. Centrifuge at 3000 RCF for 5 min, and carefully decant the supernatant
- d. Add 10 ml of PBS solution and vortex sample to suspend pellet
- e. Centrifuge at 3000 RCF for 5 min, and carefully decant the supernatant
- 8. Centrifuge Sample at 5000 rcf for 5min, decant supernatant
- 9. Cover with parafilm and poke holes in the parafilm. Place cap on over parafilm. Freeze sample in -80°C Freezer.
- 10. Start the lyophlizer cooling unit and run for 10 min prior to running.
- 11. 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.
- 12. When removing from lyophilizer, make sure the lyophilizer lid is off in order to let lyophilizer dry out.
- 13. Place 45 mg of lyophilized matter into a 50ml centrifuge tube. Label the tube.
- 14. Place 45ml of 0.9 M HCl (or load to 1mg/ml if less than 45 mg lyophilized biosolids)
- 15. Place in oven for 3h at 100°C
- 16. Cool Sample in an ice bath prior to HPLC analysis
Common Mistakes
| Mistake | Evidence of the Mistake | Cause | Effect on Results |
|---|---|---|---|
Precision and Interference
- ↑ "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.
- ↑ RedCorn, Engelberth (2018). "Quantifying Glycogen in Solids at Full-Scale Enhanced Biological Phosphorous Removal Wastewater Facilities". Journal of Environmental Engineering. 144: Issue 9.
