Buffers

0.2M sodium phosphate buffer is used for the preparation of fixative solutions and 0.1M buffer, and should never be used straight for storing specimens.

Sodium phosphate dibasic is available in several forms that vary in the amount of water incorporated in the crystal structure. It is imperative that the heptahydrate form be used with the formula above. The forms vary significantly in their molecular eight, and if other forms are used to prepare buffer, an appropriate change in the weight of reagent used must be made, otherwise both the pH and osmolarity of the buffer will be incorrect.

1.0M Sodium Phosphate Buffer: Combine 100ml of 0.2M phosphate buffer with 100ml distilled water. Add 16g anhydrous dextrose (D-glucose) and mix until dissolved. Add 1ml of 0.0002M CaCl₂ (optional).

0.1M sodium phosphate buffer is used to rinse specimens after fixation, and for short-term storage. Bacteria and fungi will grow in phosphate buffers. Stock buffers should be kept under refrigeration, and checked for contamination prior to use.

Fixatives

Glutaraldehyde can be purchased in small sealed vials with excellent storage qualities for small-volume users, or larger bottles for higher-volume users. Once mixed with buffer, the glutaraldehyde becomes less stable. The mixed fixative is usually good for a week or more if refrigerated, but should be checked carefully before use. If a white precipitate has formed, it should be discarded.

If 25% or 50% stock glutaraldehyde is used, it should first be diluted to 8% with distilled water, then combined with an equal volume of 0.2M buffer. If the glutaraldehyde is diluted to a final concentration of 4% with the 0.2M buffer alone, the osmolarity of the fixative will be too high, causing dehydration and poor preservation of the tissue.

Neutral Buffered Formalin (NBF): NBF can be obtained commercially, or made by diluting stock 37% formaldehyde 1:4 with distilled water, and mixing with an equal volume of 0.2M buffer. While glutaraldehyde fixation is preferred for most TEM applications, specimens which have been fixed promptly in formalin generally give very acceptable results.

We will be happy to supply fixatives, buffers, and specimen vials free of charge to researchers submitting specimens to our lab.

Embedding Resins

We also use Spurr's resin for specimens, such as plants, fungi, or parasites, where poor resin penetration may be a problem.

If any other type of resin embedding for TEM is required, please give us prior notice so we can make sure fresh resins are on hand.

We also routinely embed specimens for light microscopy in Immuno-Bed or JB-4 resins.