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Embedding Techniques in Tissue Histological

Chapter · January 2018

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Chief Editor
Subha Ganguly
Associate Professor, Department of Veterinary Microbiology, Arawali
Veterinary College (Affiliated to Rajasthan University of Veterinary and
Animal Sciences, Bikaner), N.H. – 52 Jaipur Road, V.P.O. Bajor, Dist.
Sikar, Rajasthan, India
Editor
Kavita Rohlan
Assistant Professor, Department of Veterinary Anatomy and Histology,
Arawali Veterinary College (Affiliated to Rajasthan University of
Veterinary and Animal Sciences, Bikaner), N.H. – 52 Jaipur Road, V.P.O.
Bajor, Dist. Sikar, Rajasthan, India
Co-Authors
Shweta Choudhary
Assistant Professor, Department of Livestock Production Management,
Arawali Veterinary College (Affiliated to Rajasthan University of
Veterinary and Animal Sciences, Bikaner), N.H. – 52 Jaipur Road, V.P.O.
Bajor, Dist. Sikar, Rajasthan, India
Vikas Kumar
Assistant Professor, Department of Veterinary Anatomy and Histology,
Arawali Veterinary College (Affiliated to Rajasthan University of
Veterinary and Animal Sciences, Bikaner), N.H. – 52 Jaipur Road, V.P.O.
Bajor, Dist. Sikar, Rajasthan, India
Nikhil Shringi
Teaching Associate VUTRC, Department of Veterinary Anatomy and
Histology, College of Veterinary and Animal Science, Rajasthan
University of Veterinary and Animal Sciences, Dist. Bikaner, Rajasthan,
Indi.

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 Chapter - 4
Embedding Techniques in Tissue Histological Process

Abstract
Embedding techniques were first developed in the mid-1800s in
response to the significant improvements in light microscopy. Edwin Klebs
introduced the paraffin-embedding methodology in 1869. As the resolution
of microscopy increased, so did the need for improved quality of the tissue
specimens to be analyzed. The specimens needed to be cut in much thinner
slices, which could only be done if embedded in a suitable medium
supporting the material and providing the hardness required for thinner
sectioning.
Keywords
Embedding, specimen, medium
Introduction
Edwin Klebs introduced the paraffin-embedding methodology in 1869.
Embedding techniques using waxes and resins have been developed and
perfected for different specific aims and different types of specimens.
Paraffin is most suitable for embedding soft tissues and decalcified hard
tissues for thin sections of 3–6μm, and is the most widely used embedding
method. Celloidin is a better option when working with large, harder, and
more fragile tissues. Depending on the size of the specimen, sections of 3–12
μm can be cut.
Hard embedding materials such as glycol methacrylate, methyl
methacrylate (MMA), or Spurr’s resin are chosen for undecalcified hard
tissue embedding suitable for heavy-duty sectioning or ground sectioning.
Sections from the latter method are relatively thick, ranging from 50–200
μm.
Before embedding, the specimens require a lengthy time for fixation,
decalcification (although not for ground sectioning), dehydration, clearing,
and impregnation or infiltration.
Each step is interdependent, and failure in one of these will directly

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affect both the ease of sectioning and the quality of the sections. Thus, it is
important that all the steps of processing be carried out by a patient, careful,
and responsible technician. More recent findings show that use of a
microwave may significantly reduce the time for fixation, dehydration,
clearing, infiltration and even staining, with no obvious adverse effects on
the tissue structure.
Paraffin Embedding and Its Processor-
Since water and paraffin do not mix, the first step in embedding with
paraffin is to replace the water in the tissues with a solvent that is miscible
with paraffin. Following are the steps in paraffin embedding:
1. Dehydration - It is the first part of the process. It is usually
accomplished by transferring the block of tissue through a series of alcohol-
water solutions beginning with 50 percent and running up to water-free or
absolute alcohol.
2. Clearing - The alcohol is replaced by Histoclear (a non-toxic
substitute for xylol) or cedar oil, which is readily soluble in alcohol, and in
turn, is replaced by melted paraffin.
3. Embedding - The actual embedding takes place when the paraffin-
infiltrated tissue is placed in fresh paraffin and the latter allowed to cool. It is
important to remember that the xylol and other solvents will dissolve the fats
of the tissues unless they are fixed by some special chemical such as osmic
acid.
Various types of embeddings are as follows-
Celloidin Embedding
Celloidin is dissolved in equal parts of absolute alcohol and ether. The
tissue is dehydrated in alcohol in the same way as for paraffin except that it
is transferred from absolute alcohol to a dilute solution of celloidin. As the
alcohol and ether evaporate, they are replaced by more concentrated
celloidin. It is finally hardened in chloroform and stored in 80 percent
alcohol. It is a much longer process than paraffin but causes much less
shrinkage and distortion. It is used especially in examination of the eye and
brain.
Epoxy Embedding
Introduction of epoxy embedding media has greatly reduced artifacts
due to shrinkage and also has allowed thinner sectioning than was possible
with paraffin. The thinner sections (approximately 1 u) may be viewed after

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staining with the light microscope or may be sectioned thinner and examined
by electron microscopy.
References
1. Blomlof L, Lindskog S, Appelgren R, et al: New attachment in
monkeys with experimental periodontitis with and without removal of the
cementum. J Clin Periodontol 14: 136–143, 1987.
2. Boon ME, Kok LP, Ouwerkerk-Noordam E: Microwave-stimulated
diffusion for fast processing of tissue: reduced dehydrating, clearing, and
impregnating times. Histopathology 10: 303–309, 1986.
3. Gruber HE, Stasky AA: Large specimen bone embedment and
cement line staining. Biotech Histochem 72: 198–201, 1997.
4. Yuehuei H. A Patricia L. Moreira Qian K. Kang Helen E. Gruber:
Principles of Embedding and Common Protocols, pp 185-197, 2003.

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