8/18/2019 Fixing Agents

1/5

Fixing agents

 There are a number of reagents that can be used to xtissues. Formaldehyde, by far the most popular agent used forhistopathology and glutaraldehyde, widely used forultrastructural studies requiring electron microscopy, aredescribed here.

Formaldehyde: Formaldehyde (CH!" is the only gaseousaldehyde and is dissol#ed in water to saturation at $%& ' )&w*#. This solution is generally referred to as +formalin or+concentrated formaldehyde solution. For xation, one partformalin is usually diluted with nine parts of water or bu-er.

 This produces a )& formalin solution which contains about& formaldehyde w*#, an optimal concentration for xation. /nconcentrated solutions formaldehyde exists as itsmonohydrate methylene glycol and as low molecular weightpolymeric hydrates. /n its diluted form the monohydratepredominates. 0araformaldehyde, a highly polymerised formof formaldehyde may be deposited as a white precipitate inconcentrated formaldehyde solutions. To pre#ent this, smallquantities of methanol (up to 1&" are commonly added to

proprietary solutions. 0araformaldehyde can be purchased asa dry powder and used to ma2e up highly pure solutions offormaldehyde such as those required for electron microscopy., $ 

3nbu-ered formalin will slowly oxidi4e to formic acid resultingin a fall in pH. 3nder these conditions the formic acid will reactwith hemoglobin forming acid formaldehyde hematin, abrown5blac2 granular artefact pigment which is deposited inblood5rich tissues. This pigment is a nuisance as it can be

confused with micro organisms or other pathologicalpigments.  6lthough the pigment can be remo#ed fromsections with saturated aqueous picric acid before staining, itis preferable to a#oid its formation in the rst place. For thisreason and because formaldehyde reacts most e-ecti#ely atabout a neutral pH, )& formalin solutions are usuallybu-ered to pH 7.8 ' %..

http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/#c22470http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/#c22470http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/#c22470http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/#c22470

8/18/2019 Fixing Agents

2/5

Figure 9 6 formalin5xed para:n section of 2idney showingthe typical deposition of acid formaldehyde hematin (formalinpigment" associated with red blood cells. The pigment isbrown to blac2 in color and is birefringent under polari4ed

light. /n this case the specimen remained in xati#e for anextended period before processing.

Formaldehyde reacts with the side5chains of proteins to formreacti#e hydroxy5methyl groups. /t can penetrate nuclearproteins and nucleic acids stabili4ing the nucleic acid5proteinshell and modifying the nucleotides by reacting with freeamino groups. Formaldehyde can react with some groups inunsaturated lipids particularly if calcium ions are present, buttends to be unreacti#e with carbohydrates. 1 Formaldehydecan react with groups on lysine, arginine, cysteine, tyrosine,threonine, serine and glutamine forming reacti#e complexeswhich may combine with each other forming methylenebridges (cross5lin2s" or with hydrogen groups. 1 /t is widelyaccepted that washing tissues after formalin xation canre#erse some of these reactions but important cross5lin2s

http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/#c22470http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/#c22470http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/#c22470http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/#c22470

8/18/2019 Fixing Agents

3/5

remain. 7 /t is the ability of formaldehyde to preser#e thepeptides of cellular proteins which ha#e made it so useful as ageneral purpose xati#e.

 There are well 2nown ha4ards associated with the use offormaldehyde as a xati#e through s2in or eye contact or #iathe respiratory tract. /t is irritant, corrosi#e and may causeallergic sensiti4ation. From ;8 formaldehyde was listed as+reasonably anticipated to be a human carcinogen and in) the listing has been upgraded to +2nown to be a humancarcinogen.%, 8 

8/18/2019 Fixing Agents

4/5

groups as formaldehyde. They will form addition compoundsand methylene bridges but also a single glutaraldehydemolecule may form direct cross lin2s if the steric arrangementof adAacent peptides allow it. The amino groups of lysine areparticularly important in this respect. Tissue xed inglutaraldehyde will be more extensi#ely cross5lin2ed thantissue xed in formalin and will also possess some unreactedaldehyde groups that, unless chemically bloc2ed, can causebac2ground staining in methods such as 06

8/18/2019 Fixing Agents

5/5

Figure $9 6n electron micrograph showing a anterman incisure in a myelinated ner#e bre. This specimenwas xed in bu-ered glutaraldehyde, washed in bu-er thengi#en secondary xation in bu-ered osmium tetroxide, astandard procedure when preparing specimens fortransmission electron microscopy. The multiple layers ofphospholipid membrane forming the myelin sheath are well5preser#ed by this procedure.