Measurement of Glycogen in Microbiota

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

1. Label Tubes;
2. 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.
3. Place aliquots of 40 ml per sample in 50 ml centrifuge tubes.
4. Collect sludge in a 15 ml centrifuge tube (should be about 20 mg solids worth) through one of the following methods;
5. Store at 4°C for 2 hours
6. 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
7. Cover with parafilm and poke holes in the parafilm. Place cap on over parafilm. Freeze sample in -80°C Freezer.
8. Start the lyophlizer cooling unit and run for 10 min prior to running.
9. 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.
10. When removing from lyophilizer, make sure the lyophilizer lid is off in order to let lyophilizer dry out.
11. Place 45 mg of lyophilized matter into a 50ml centrifuge tube. Label the tube.
12. Place 45ml of  0.9 M HCl (or load to 1mg/ml if less than 45 mg lyophilized biosolids)
13. Place in oven for 3h at 100°C
14. 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