Understanding Histological Techniques for Tissue Preparation
Histological technique involves preparing tissue for microscopic examination, aiming to preserve the microscopic anatomy and structure of tissues. The process includes fixation, dehydration, cleaning, embedding, cutting, and staining. Key steps like fixation using fixative materials help prevent autolysis, preserve tissue integrity, and make it suitable for staining, ultimately allowing for detailed microscopic analysis.
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INTRODUCTION Histological technique deals with the preparation of tissue Histological technique deals with the preparation of tissue for microscopic examination. for microscopic examination. The aim of good histological technique to preserve The aim of good histological technique to preserve microscopic anatomy of tissue. microscopic anatomy of tissue. Make them hard so that very thin section ( Make them hard so that very thin section (4 4 to can be made. can be made. Good staining should be possible. Good staining should be possible. to 5 5 micron) micron) After staining, After staining, the section should represent the anatomy of the section should represent the anatomy of the tissue as close to as possible to their structure in the tissue as close to as possible to their structure in life. life. This is achieved by passing the total as selected part of This is achieved by passing the total as selected part of the tissue through a series of process. the tissue through a series of process.
SPECIMENS IN HISTOLOGY Biopsy: piece of tissue or organ taken from living human being. Autopsy: piece of tissue or organ taken from dead body. Ex: blood, CSF, ascites fluid, pleural effusion fluid. * How to study the specimen: 1. Gross: colour, size, surface, texture, consistency. 2. Microscopical examination: examine under microscope.
TECHNIQUES These techniques are: These techniques are: 1 1. Fixation . Fixation 2 2. Dehydration . Dehydration 3 3. Cleaning . Cleaning 4 4. Embedding . Embedding 5 5. Cutting . Cutting 6 6. Staining . Staining
FIXATION FIXATION This is the process by which cells and This is the process by which cells and tissue are fixed in a physical and chemical tissue are fixed in a physical and chemical state so that they will withstand subsequent state so that they will withstand subsequent treatment with various reagents with treatment with various reagents with minimum loss of architecture minimum loss of architecture . . This should be approximately This should be approximately 10 volume of the specimen. Fixative should volume of the specimen. Fixative should surround the specimen on all sides. surround the specimen on all sides. 10- -20 20 times the times the
BASIC STEPS FOR PREPARATION OF STAINED SECTION FROM TISSUE 1. Fixation: the aim of this step are: To prevent autolysis To preserve tissue as nearly as possible to living state. To prevent damage during subsequent procedures To give a suitable texture. To prevent growth of bacteria. To render tissue receptive to subsequent staining.
Fixation is done by using fixative materials like: 1. Formaline 10%. 2. Alcohol 75%. 3. Zenker s fluid. 4. Bouins fluid: it provide balance between those ingredients that cause shrinkage of the cell and thiose cause sweelling of the cell. 5. Carnoy s fluid.
It requires It requires 24 It can be subdivided into It can be subdivided into a) dehydration a) dehydration b) clearing b) clearing c) impregnating c) impregnating d) embedding. d) embedding. Note: Note: It is important that all specimens are properly labeled It is important that all specimens are properly labeled before processing is started. before processing is started. 24hours and done in many stages. hours and done in many stages.
SEQUENCE OF TISSUE PROCESSING SEQUENCE OF TISSUE PROCESSING Dehydration Dehydration: :Tissues dehydration water in tissue has been dehydration water in tissue has been replaced by alcohol. The next step alcohol should be replaced by alcohol. The next step alcohol should be replaced by paraffin wax. replaced by paraffin wax. Clearing of tissue is achieved by any of the following Clearing of tissue is achieved by any of the following reagents:Xylene reagents:Xylene, , Chloroform,Benzene Chloroform,Benzene Note: Note:Xylene Xylene is commonly used. is commonly used. a. Xylol: cheap, rapid and tends to harden tissue on prolong application. b. Choloroform: makes tissue less brittle than xylol it cause shrinkage on prolong use. . . Tissues are dehydrated are dehydrated Clearing: Clearing:During During
2. Dehydration: this process is done to pull the water from sample, and carried out by using alcohol in various dilutions(by increasing increasing strength strength of of alcohol alcohol; ; e e. .g g. . 90 90% % and and 100 100% %. .) ). Acetone is another agent can be used. by using using 50 50% %, , 70 70% %, , 3. Clearing: done by: a. Xylol: cheap, rapid and tends to harden tissue on prolong application. b. Choloroform: makes tissue less brittle than xylol it cause shrinkage on prolong use.
Embedding Embedding Impregnated tissues are placed in a Impregnated tissues are placed in a mould and then fresh melted wax is poured in it and allowed to and then fresh melted wax is poured in it and allowed to settle and solidify. settle and solidify. Once the block has cooled sufficiently to form a surface Once the block has cooled sufficiently to form a surface skin it should be immersed in cold water to cool it rapidly. skin it should be immersed in cold water to cool it rapidly. . . Microtomy Microtomy For For light microscopy light microscopy emotorcim a ni detnuom efink ssalg a , emotorcim a ni detnuom efink ssalg a , tuc ot desu si tuc ot desu si4 4- -6 6 um um- -thick tissue sections which are thick tissue sections which are mounted on a glass microscope slide mounted on a glass microscope slide . mould with their labels with their labels
STAINING STAINING Staining is a process by which we give color to a section. Staining is a process by which we give color to a section. There are hundreds of stains available. There are hundreds of stains available. The stains can be hematoxylin/eosin stain or special stains The hematoxylin/eosin stain is usually abbreviated as h&e stain. The H&E stain is routinely used. It gives the nucleus a blue color & the cytoplasm & the extracellular matrix a pinkish color.
4. Impregnation: This is allowed to occur at melting point This is allowed to occur at melting point temperature of paraffin wax, which is temperature of paraffin wax, which is 54 should be about should be about 25 25- -30 30 times the volume of tissues. times the volume of tissues. 54- -60 60oC. Volume of wax oC. Volume of wax tissue impregnated with wax for two reasons: a. To surround tissue with substance to support it to put on slide without injury. b. To enable natural cavities of tissue to be filled with wax thus preserving their relationship to each other.
. 5. Adhesion to section slide: this is usually done by smearing with egg albumin. 6. Drying: put the slide for (10-15) min in oven at 65C.
STEPS OF STAINING WITH HEMATOXYLIN-EOSIN (HE) 1. Xylene wait for 1 min to remove the substance that are not embedded in slide. 2. Alcohol 70% wait for 1 min. 3. 3. Alcohol 90% wait for 1 min. 4. Alcohol absolute wait for 1 min. 5. Hematoxylin wait for 5-10 min. 6. Wash with tab water. 7. Eosin wait for 1-2 min. 8. Wash with tab water. 9. Dry by using oven. 10. Clear by xylene and then using Canada balsam.
FROZEN SECTION BIOPSY This is used for the spot diagnosis, usually fresh unfixed tissue is utilized. Afreezing microtome or cryostat machine at a tempreture of -20 to -30 is used, the section is cut at thickness of 5-10 microns then staining can be used for these sections. .
DECALCIFICATION OF BONE AND OTHER TISSUE Sometimes its necessary to remove deposits of calcium from tissues e.g.. Calcified arteries, bones, teeth etc. this should be done immediately after fixation, fixatives containing mercuric chloride cause swelling of soft tissue during decalcification. If large piece is submitted: cut into small pieces and then suspend the tissue in decalcification fluid from these fluids: - Normal saline - Nitric acid materials.
