Lecture (4) commonly used fixatives in the laboratory

Commonly used fixative in the laboratory

1. Commonly Used Fixatives in the Laboratory By: Ms. Hafsa Sh. Hussein Histopathology & Cytopathology Lecturer Faculty of Laboratory, EAU. Lecture 4:

2. Bouin’s Fixative

3. Contains picric acid and Formaldehyde . Excellent for glycogen . Reacts with protein and forms protein picrate . Penetration rate is high . it fixes small tissue within 3–4 h. Bouin’s fixative is not suitable for DNA study damages the cell membrane hydrolysis of nuclei acid.

4. Advantages : Good for connective tissue & glycogen . Rapid penetration rate.

5. Yellow stain on the tissue. Removal of yellow colour : Removed by dipping the tissue in lithium carbonate in 70% alcohol. Disadvantages :

6. Mercury Salt – Containing Fixatives

7. Zenker’s fluid Helly’s fluid B5 fixative

8. This is a rapidly acting fixative. Contains mercury (heavy metals) Mercury is a poisonous substance and should be used carefully . Mercury containing fixatives may corrode the metal so the fixative should be kept in glass container .

9. Contains Mercuric chloride , Potassium dichromate and Glacial acetic acid . It is a good fixative for: nuclear chromatin collagen . Organ with very high vascularity such as the spleen Zenker’s fluid

10. Helly’s Fluid This is a good cytoplasmic fixative. Bone marrow Rarely used. Contains Mercuric chloride and formaldehyde . It takes nearly about for 3 mm/12 h for tissue to be fixed.

11. B5 Fixatives Used for bone marrow biopsy and lymph node. Contains Mercuric chloride and formaldehyde .

12. Fixation Artefact

13. Causes : Unbuffered formalin is kept for long time. It is converted into formic acid Formic acid + haemoglobin = Acid formaldehyde haematein Appearance: produce insoluble brownish-black granular pigment . A. Formalin Pigment

14. The section is immersed in xylene Followed by alcohol to bring in water. Subsequently the section is immersed in 1.8% picric acid in absolute ethyl alcohol for 15 min . It is then washed thoroughly. Section is re-stained. Removing the pigment:

15. Causes: When tissue is fixed by mercuric chloride Appearance: Produce a dark-brown irregular deposit. Removal: Application of iodine converts it into mercuric iodide which is removed by sodium thiosulphate . B. Mercury Pigments :

16. Causes: Improper fixation either due to: Insufficient fixative. Too little time in fixative. Appearance: The nuclear and cytoplasmic details are obscured and the section looks fuzzy or hazy. C. Fuzzy Staining:

17. Causes: Fixation time is too Long Appearance: Shrinkage of the tissue. Separation of the tissue. Show holes or empty spaces within the tissue. D. Prolonged fixation :

18. Causes: Chromium salt may formation Tissue is not properly washed after dichromate fixation. Appearance: insoluble yellow-brown precipitate Chromium salt + alcohol = insoluble yellow-brown precipitate . E. Dichromate deposit :

19. Removal: 1% hydrochloric acid in 70% alcohol for 30 min

20. Books: " Basic and Advanced Laboratory Techniques in Histopathology and Cytology " by  Pranab Dey