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1. Mounting in Histopathology

2. Outline Mounting (Introduction) Mounting Media Mounting Techniques

3. Mounting

4. Mounting Arranging tissue section on a slide before it is covered with cover slip is known as mounting . Adhesive used to fix (adjust) tissue section on slide is known as mountant or mounting media . A sufficiently thin & suitably colored slice of biological material must be placed on glass slides for examination under microscope. A drop of mounting media is applied to sections, & coverslip is carefully dropped onto sample using tweezers. This process is called mounting in histopathology .

5. Purpose of Mounting To protect specimen from physical harm To protect section from bleaching or degradation due to oxidation To preserve slides for long-term storage To allow simple handling & storage To avoid section damage

6. Mounting Media

7. Mounting Media Medium in which your sample is in while being imaged by microscope. or Medium in which your sample is embedded under coverslip. Mounting media can be adhesive or a syrup solution. Common adhesives used are: 1% Gelatin Mayer’s albumin Dried Albumin

8. Mounting Media Adhesives are used particularly when exposure of tissue sections to acids or alkalis are required during staining. They are smeared to glass slides , so tissue sections stick to glass slides. However, mostly syrup solutions are applied between tissue section & cover slip after staining. For such medium, slides are taken from last xylene bath using forceps. Then a drop of mounting media is applied & coverslip is placed. After that slide is incubated at 37 degree Celsius to harden medium, if needed.

9. Characteristics of Good Mounting Media Refractive index of mounting media should be near to 1.518 . Should be Freely miscible with xylene & toluene . Should not dry quickly . Should not crack or produce artifactual granularity of slide upon drying. Should not dissolve out or fade tissue sections. Should not cause shrinkage or distortion of tissue sections. Should not bleach out or affect staining . It should not change color or pH .

10. Types of Mounting Media Aqueous Media Resinous Media Specialized Mounting Media

11. Aqueous Media: It includes such solution or substances which are designed to mount water solvent preparations directly from water in case where stain removed from alcohol or xylene. Examples: Water (has low refractive index but doesn't allow tissues to be examined under 100X lens, only temporary mounting). Glycerin (Has R.I of 1.46 , suitable for semi permanent mounting, it can keep tissue sections preserved for long time). Apathy’s medium (Used for methylene blue stained nerve preparations).

12. Resinous Mounting: Used for preparations that have been dehydrated & cleared in xylene or toluene & are recommended for majority of staining methods. They are divided into two categories:- Natural (Canada balsam) Synthetic DPX (Dibutyl phthalate & xylene) R.I = 1.52 Histomount (R.I = 1.49-1.50).

13. Specialized Mounting Media Fluoromount: Used for fluorescent samples, it reduces photobleaching. Example: Mounting fluorescently labeled cells. Vectashield: Also for fluorescence, available with or without DAPI (a DNA stain). Example: Mounting DAPI-stained chromosomes. Hoyer’s Medium: Used for mounting small invertebrates, particularly in entomology. Example: Mounting mite specimens.

14. Different Mounting Techniques

15. Types of Mounting Techniques Dry Mounting Wet mounting Prepared/Ready Mounting

16. Dry Mounting

17. Dry Mounting Mounting where specimen is directly placed on slide without use of any liquid medium. In this method, specimen is typically dry, & no mounting medium or cover slip is added on top. Commonly used for observing dry & solid samples or specimens that do not require additional preparation or staining. Dry mount slides are useful for: Solid chemicals & crystals Textiles & fibers Pollen grains & plant parts Dust particles Insect parts (e.g., wings, legs) Feathers & scales Dry food particles (e.g. spices, sugar crystals) Non-living biological materials Hair & fur samples

18. Advantages of Dry Mounting Dry Mounting is quick & easy as it is a rapid & straight forward process requiring minimum materials & steps. Dry mounting involves direct observation of objects as no liquid medium is used. Dry mounting is cost effective & requires low power microscopy. Dry mounting involves variety of objects which can be examined & structural features remain preserved.

19. Limitations of Dry Mounting Absence of a mounting medium with a specific refractive index can result in lower resolution & clarity of specimen under microscope. Without a medium to optimize light refraction, image may appear darker & less detailed . Dry-mounted slides lack support provided by a surrounding mounting medium. As a result, they are more prone to movement & shifting of specimen under cover glass. Some specimens may undergo shrinkage or deformation during drying process, leading to altered structures & distorted observations . This can particularly affect delicate or organic materials.

20. Wet Mounting

21. Wet Mounting Used to view liquids under microscope. Preparation of a wet mount slide includes a depression slide, a cover slip, & an eye dropper can be useful, but is not required. Wet mount slide samples might include pond water, cheek cells, blood or sperm samples . When preparing wet mount slide, place a small drop of the sample between depression slide & cover slip. Use a paper towel when pressing cover slip onto slide, as some of your sample might run out over edges.

22. Common Problems Air Bubbles Formation: Air bubbles can form if your slide coverslip closes too rapidly causing air to be trapped under coverslip or a displacement in the water that is rapidly filled by air. Contamination: Contamination can occur if you add contaminated water to your slide that contains other microorganisms or even chemicals that may affect you intended specimen. To avoid contamination, you need to understand your specimen & what things in your water source may cause contamination or unintended consequences.

23. Coverslip Floating: In some cases, you may find that if you add too much water your coverslip will float after you place it over the water droplet. This can cause your slide coverslip to move to one side & can also disrupt your sample in some cases. Coverslip floating can be avoided by limiting amount of water that is dropped onto slide & by slowly lowering coverslip on slide.

24. Ready / Prepared Mounting Making a prepared mount slide is more complicated, although it is more long-lasting than slides prepared using first two techniques. Specimen must be sliced very thinly . Simplest tool used for this is often a microtome . Once specimen is thin enough, it is carefully placed on glass slide. Any excess water is needed to be removed before adding a suitable dye so as to stain intricate structures of specimen. After washing excess stain away, coverslip is placed on top of sample. Adding a fixative to specimen before you set on coverslip will help prevent decay.

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