Can anyone suggest me whole procedure of preparing 4% glutaraldehyde for fixation of samples required for SEM studies.We have glutaraldehyde Grade I, 70% in water. For preparing above solution, we need phosphate buffer solution and that is to be prepared fromNaH2PO4 * H2O and Na2HPO4 (anhydrous) salts. How phosphate buffer solution required for preparing gutaraldehyde is to be prepared from above to salts. Please provide whole procedure?
Dear Aashaq,
4% glutaraldehyde solution is 4 g glutaraldehyde in 100 mL solvent. Since your Glut. is 70% to make 4 gram you need to use 5.56 gram (70% glutaealdehyde Grade I).
Phosphate Buffer; prepare 0.2 M phosphate buffer by using NaH2PO4 * H2O and Na2HPO4, adjust the pH with NaOH or HCl.
Procedure:
1-add 5.56 gram 70% glutaealdehyde Grade I to 50 mL distilled water.
2-Make up to 100 mL with 0.2 M phosphate buffer.
Preparation of 0.2 M Phosphate Buffer
A (mL)..........B (mL)..........pH
23................77................7.3
19.................81................7.4
16................84................7.5
Solution A: NaH2PO4.2H2O, mol wt 156.01, 3.12 g in 100 mL H2O.
Solution B: Na2HPO4, mol wt 141.96, 2.84 g in 100 mL H2O.
Note: pH 7.4 buffer fits most of samples.
Buffer is made from the phosphate salts indicated in your question.
Hoping this will be helpful,
Rafik
Dear Aashaq,
4% glutaraldehyde solution is 4 g glutaraldehyde in 100 mL solvent. Since your Glut. is 70% to make 4 gram you need to use 5.56 gram (70% glutaealdehyde Grade I).
Phosphate Buffer; prepare 0.2 M phosphate buffer by using NaH2PO4 * H2O and Na2HPO4, adjust the pH with NaOH or HCl.
Procedure:
1-add 5.56 gram 70% glutaealdehyde Grade I to 50 mL distilled water.
2-Make up to 100 mL with 0.2 M phosphate buffer.
Preparation of 0.2 M Phosphate Buffer
A (mL)..........B (mL)..........pH
23................77................7.3
19.................81................7.4
16................84................7.5
Solution A: NaH2PO4.2H2O, mol wt 156.01, 3.12 g in 100 mL H2O.
Solution B: Na2HPO4, mol wt 141.96, 2.84 g in 100 mL H2O.
Note: pH 7.4 buffer fits most of samples.
Buffer is made from the phosphate salts indicated in your question.
Hoping this will be helpful,
Rafik
Thanks Rafik Karaman Sir for your response but the glutaraldehyde we have is in liquid form not in crystal form so how much ml of glutaraldehyde Grade I, 70% in water is required to prepare 4% glutaraldehyde using phosphate buffer.
Dear Aashaq,
it does not matter. you can weigh liquids. Otherwise: weight = volume x density;
Volume = weight/density; divide 5.56 g with the density you get the number of mL.
Rafik
Do you recommend this fixation method to examine liposomes under a SEM?
Dear David E. Bautista-Erazo
You can try the above method to see good results. I recommend you to read the paper I attached with this for more details.
Regards
AH Bhat
Did you use malachite green with good results? I read that paper before and I wanted to be sure that it's going to work. If you tested it, do you have any tips or recommendations? Thanks.
No Omprakash Ghimire, SEM is Scanning Electron Microscopy, it only allows you to scan the surface of an object. If you need to look through the cell you should try TEM (Transmission Electron Microscopy)
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