Define Fixative types and classification

Fixative and its classification and there type

1. Fixatives M. Imran khan Lecturer MLT NWIHS

2. 1- Properties of Formal dehyde Belongs to the class of Aldeyde . Basically in gaseous form and solubility in water is 37-40%. This aqueous solution of formaldehyde is named as formalin and is commercially available. This formalin solution is considered as 100% and then 10 % solution is prepared for fixation. Merits: 1. Comparatively cheaper so is used in busy labs. 2. Easy to prepare.

3. 3. Fix tissue in their natural color that’s why it is used as museum fixative. 4. Penetration is adequate. 5. Shrinkage and brittleness are not caused by formal dehyde . 6. Surgical specimens rapidly fixed depending on their size. 7. Very good fixative for proteins and more over substances like lipids and mucins well preserved. 8. A multitude of staining techniques can be used on formal dehyde fixed tissues with excellent results.

4. Properties of Formal dehyde Demerits: 1. It is toxic 2. Irritant to skin so prolonged contact with formal dehyde may cause dermatitis and also irritant to nasal mucosa so may cause sinusitis. 3. Spontaneous formation of formic acid in formal dehyde fixative that changes the pH due to which a brown pigment appears in tissue that impairs final staining results.

5. Properties of Formal dehyde To avoid this an alkali or basic salt is added in formal dehyde for neutrality. 4. When kept for prolonged period develop a white precipitate that is a polymer of formal dehyde . It however does not change fixing properties of fixative that can be filter or kept at higher temp to remove this polymer.

6. 2- Properties of Mercuric chloride Belongs to the class of unknown mechanism. It is a metallic salt having 7% solubility in water. Merits: 1. Mercuric chloride fixative mainly used as secondary fixative that means sometimes formal dehyde fixed tissue are again fixed in a mercuric chloride fixative at the 2 nd stage in order to enhance final staining.

7. Properties of Mercuric chloride 2. Results of H/E staining are excellent. 3. Act as a mordant due to metallic mercury i.e. staining reaction is excellent to give brilliant staining result. 4. Some special stains like trichrome staining, metachromatic staining also show good results on mercuric chloride fixed.

8. Demerits: 1 . Toxic and corrosive. 2. Mercurial's are not much as stable as formal dehyde and is easily deteriorated. This property named as shelf life. 3. A black precipitate of mercury appears in tissues fixed in mercurial that needs to be removed before further processing of tissue. 4. Cause shrinkage of tissues

9. 3- Properties of Osmium tetra oxide Belongs to the class of oxidizing agents. Solubility in water is about 6%. Aqueous solution of osmium tetra oxide is very strong oxidizing agent so very reactive, therefore must be kept in a clean dark colored bottle. Merits: 1. Good fixative for lipids as it permanently fix lipids.

10. Properties of Osmium tetra oxide 2. Cytoplasmic organelles like mitochondria, Golgi bodies are excellently fixed. 3. Vapors of osmium tetra oxide also used for fixation. Demerits: 1. More expensive

11. 2. Vapors of lower oxides like osmium dioxide are dangerous for the workers and may deposit in cornea of eye causing blindness. So the chemical must be used in well-ventilated labs. 3. Tissues fixed in oso4 needs washing out . These are washed out under running tap water for hours before taking them to reducing alcohol.

12. 4- Properties of Picric acid Belongs to class of fixatives of unknown mechanism. Solubility is about 1% in water and saturated solution is used for fixation. Dry picric acid is explosive. Merits: 1. Picric acid is a yellow color compound and gives a yellow colored solution. Tissues fixed in picric acid also yellow colored.

13. Properties of Picric acid 2. Sometimes yellow coloration is a merit as in case of tiny biopsies. Due to yellow coloration these biopsies can easily be located and handle & there is less chance of loosing during performance of different procedures. 3. Recommended for glycogen histochemistry (study of different chemicals in tissues).

14. Demerits: 1. Sometimes this yellow coloration is demerit b/c it impairs final staining and several washings are required in alcohol or xylene to remove the yellow color.

15. 5- Properties of Glacial acetic acid Belongs to the class of protein denaturing agent. Merits: 1. Good fixative for chromatin material so is widely used when chromosomal studies are required. 2. Cause swelling of tissues so is used in combination with such fixatives that cause shrinkage so that the overall effect in the volume of tissue is balanced.

16. Properties of Glacial acetic acid 3. Cause lyses of RBC’s, sometimes it is required to fix tissues containing a lot of blood in order to remove excess blood and study details of other cells. 4. It is quick in action so fixing tissues comparatively in shorter time therefore it is used in high work load labs or in case of some emergency.

17. Demerits: 1. Biopsies like bone marrow should never be fixed in GAA in order to preserve RBC series cells. Bone marrow is best fixed in zenker formol . 2. Cause lyses of many cytoplasmic organelles like mitochondria, Golgi bodies. 3. Causes swelling of tissues.

18. 6- Properties of potassium dichromate Belongs to the class of Oxidizing agent. 3% aqueous used as a fixative. Merits: 1. To fix certain lipids specially in the myelin sheath. This property is lost if the fixative is mixed with glacial acetic acid.

19. Properties of potassium dichromate 2. Potassium dichromate fixation gives enhanced subsequent staining, due to this property sometimes tissues fixed in formal dehyde are kept for sometime in potassium dichromate solution. ( secondary fixation ). Demerits: 1. P. dichromate can not be used in combination with GAA b/c lipids fixing property of P.dichromate destroyed.

20. 2. Tissues fixed in P.D needs washing out. 3. Highly reactive so it should be carefully kept in clean container free of any organic materials. 4. In secondary fixation , formal dehyde fixed tissue are kept in p.dichromate for about 3 hrs. This treatment is named as post chroming or post chromatization , it is considered that this treatment enhance final staining.

21. 7- Properties of Ethanol Belongs to the class of protein denaturing agent. Merits: 1. non toxic 2. Rarely used as simple fixative as in case of glycogen demonstration where ever it is an important component of compound fixative. Demerits: 1. very expensive having high excise duties. 2. Causes considerable shrinkage and hardness in tissues that is why it is always used in graded strength.

22. Factors affecting fixation Temperature: Rate of fixation is increased with rise in temperature that’s why for emergency biopsies heated formalin is used (at 60 °C for 1 hr). However with rise in temp the process of autolysis also increases so fixatives are usually used at room temp.

23. Factors affecting fixation Time: Tissue must be fixed for proper time depending on the type of fixative used. Inadequate time give poor fixation while keeping tissue in a fixative more than the required time may make it very hard & brittle.

24. Factors affecting fixation Penetration of fixative: Different fixative show different penetration. The penetration of fixative is given by its coefficient of diffusibility (K) It is defined as, penetration of fixative in mm/hr. Example : K Formal dehyde 0.78 Picric acid 0.5 Ethanol 1.0 Potassium dichromate 1.33 K=d/t = Penetration/time = mm/hr

25. Factors affecting fixation Concentration: Rate of reaction increase if concentration increase. Concentration of fixative show certain limits like cost, solubility and effectiveness. More expensive fixative are used in lower conc and vice versa. Conc is also limited by solubility. e.g. 1% soluble picric acid can not be used in higher conc.

26. Sometime low concentration are as effective as higher conc. e.g. 0.25 % glutar aldehyde is as effective as 3%. pH: pH also affect fixation. Most of the fixative work at acidic pH (5-5.6) if pH value changes then ionization molecules changes.

27. Secondary fixation Formal dehyde fixative mainly used for routine work so formal dehyde fixative are used as primary fixative. However sometimes formal dehyde fixed tissue are kept in fixatives like mercuric chloride. P. dichromate and osmium tetra oxide for sometime in order to enhance staining intensity on these tissues. This treatment is known as secondary fixation and fixative used are called as secondary fixative.

28. Post chromatization Sometimes formal dehyde fixed tissues are kept in 3% P. dichromate solution. It is b/c final staining results are enhanced by this treatment. It is called as post chroming or post chromatization . However post chroming needs washing out that is time consuming.

29. Washing out When tissues are fixed in an oxidizing agent these need to be washed in running tap water for hrs in order to remove the oxidizing agent from tissue and the treatment is known as washing out. It is required b/c after fixation tissues are passed through dehydrating alcohol during processing. As dehydrating alcohols are reducing agents which may react with oxidizing agent & impair the tissue.

30. Fixative artifact Fixatives are used to preserve tissue against post mortem changes but these themselves are associated with certain problems and these are collectively named as fixative artifacts. The most important artifacts are: 1. Volume changes 2. Color changes

31. 3. Deposition of pigment such as formal dehyde give brown pigment. 4. Streaming artifact: Due to slow penetration of fixative unfixed molecules in tissues diffused about giving false localization.

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