Measurement of Glycogen in Microbiota - Revision history

Raymond RedCorn at 10:29, 4 March 2019

2019-03-04T10:29:05Z

Raymond RedCorn at 10:22, 4 March 2019

2019-03-04T10:22:53Z

Raymond RedCorn at 10:22, 4 March 2019

2019-03-04T10:22:19Z

Raymond RedCorn at 04:03, 1 March 2019

2019-03-01T04:03:03Z

Raymond RedCorn: /* Materials and Equipment */

2019-03-01T04:01:46Z

Materials and Equipment

Raymond RedCorn: /* Reagents */

2019-03-01T04:01:21Z

Reagents

Raymond RedCorn: /* Precision and Interference */

2019-03-01T03:50:18Z

Precision and Interference

Raymond RedCorn at 03:45, 1 March 2019

2019-03-01T03:45:19Z

Raymond RedCorn at 03:41, 1 March 2019

2019-03-01T03:41:20Z

Raymond RedCorn: Created page with "

@@ Line 4: / Line 4: @@
 <!-- This shows the authors that have edited a page and gives them credit for their contribution -->
 ===={{Template:CiteAuthors}} ====
 ==== '''This method is also called:''' ====

@@ Line 5: / Line 5: @@
 ===={{Template:CiteAuthors}} ====
-<youtube>Hd-iV1-XCc</youtube>
+<youtube>_Hd-iV1-XCc</youtube>
 ==== '''This method is also called:''' ====

@@ Line 4: / Line 4: @@
 <!-- This shows the authors that have edited a page and gives them credit for their contribution -->
 ===={{Template:CiteAuthors}} ====
 ==== '''This method is also called:''' ====

@@ Line 69: / Line 69: @@
 ! Effect on Results
 |-
-| Leave valve to lyophilzer jar
+| Leave valve to lyophilzer jar open
-| Cooling coils on lyophilizer have excessive ice<!-- (How can you tell if the mistake has been made?)(How can you tell if the mistake has been made?) -->
+| Excessive ice forms on the cooling coils of the lyophilizer.<!-- (How can you tell if the mistake has been made?)(How can you tell if the mistake has been made?) -->
 |Moisture from the air allowed to enter. <!-- What is fundametnally happening
   -->
-| <!-- (Higher, lower, flatline?) -->Cannot complete lyophilization
+| <!-- (Higher, lower, flatline?) -->Cannot complete lyophilization in reasonable time.
 |}
 ==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<ref name=":0" /><references />

@@ Line 15: / Line 15: @@
 == Materials and Equipment ==<!-- Any tubes, pipette tips ect. -->
 * Fume Hood
 * Lyophilizer

@@ Line 32: / Line 32: @@
 | 50-00-0<!-- Universal Identifier, regardless of manufacturer -->
 |-
-|PBS Solution
+|[[Phosphate Buffered Saline (PBS) Solution|PBS Solution]]
+|
+|

@@ Line 9: / Line 9: @@
 ==== '''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.-->
-Samples can be prepped by preserving with formaldehyde prior to lyophilizing the same as methods measuring PHA.
+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. -->
@@ Line 76: / Line 76: @@
 ==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 />

@@ Line 49: / Line 49: @@
 # 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.
+# 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
-##  Centrifuge at 3000 RCF for 5 min, and carefully decant the supernatant
+# Cover with parafilm and poke holes in the parafilm. Place cap on over parafilm. Freeze sample in -80°C Freezer.
-# b.      Add 10 ml of PBS solution and vortex sample to suspend pellet
+# Start the lyophlizer cooling unit and run for 10 min prior to running.
-# c.       Centrifuge at 3000 RCF for 5 min, and carefully decant the supernatant
+# 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.
-# d.      Add 10 ml of PBS solution and vortex sample to suspend pellet
+# When removing from lyophilizer, make sure the lyophilizer lid is off in order to let lyophilizer dry out.
-# e.       Centrifuge at 3000 RCF for 5 min, and carefully decant the supernatant
+# Place 45 mg of lyophilized matter into a 50ml centrifuge tube. Label the tube.
-# 8.      Centrifuge Sample at 5000 rcf for 5min, decant supernatant
+# Place 45ml of  0.9 M HCl (or load to 1mg/ml if less than 45 mg lyophilized biosolids)
-# 9.      Cover with parafilm and poke holes in the parafilm. Place cap on over parafilm. Freeze sample in -80°C Freezer.
+# Place in oven for 3h at 100°C
-# 10.  Start the lyophlizer cooling unit and run for 10 min prior to running.
+# 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==
@@ Line 74: / Line 68: @@
 ! Effect on Results
 |-
+| Leave valve to lyophilzer jar
-| <!-- (How can you tell if the mistake has been made?)(How can you tell if the mistake has been made?) -->
+| Cooling coils on lyophilizer have excessive ice<!-- (How can you tell if the mistake has been made?)(How can you tell if the mistake has been made?) -->
-|<!-- What is fundametnally happening
+|Moisture from the air allowed to enter. <!-- What is fundametnally happening
   -->
-| <!-- (Higher, lower, flatline?) -->
+| <!-- (Higher, lower, flatline?) -->Cannot complete lyophilization
 |}
 ==Precision and Interference==

@@ Line 7: / Line 7: @@
 ==== '''This method is also called:''' ====
-==== '''This method is adapted from: <!-- Citation -->''' ====
+==== '''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>  ====
 == Discussion ==<!-- Discuss the purpose, the suitability of the method for certain situations, and the fundamentals science behind the method.-->
 == Materials and Equipment ==<!-- Any tubes, pipette tips ect. -->
 ==Reagents==<!-- Test kits go here if used-->
@@ Line 21: / Line 27: @@
 ! 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 -->
 |}
 ==Procedure==
 ==Common Mistakes==