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The Steps for the Preparation of Slides for Microscopic Examination

 

 

 

 Histology Steps

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Fixation/Processing

     
The specimens are delivered to the Histology Laboratory immediately after the autopsy. The tissue is labeled with an identifying Medical Examiner’s case number and is placed in perforated plastic capsules. The capsules are placed in a 10% formalin fixative solution. This fixes or preserves the tissue to prevent decomposition. If the tissue is not fixed properly, then appearance of the cells change. There is no way to reverse decomposition.

This step takes place in a series of solutions which wash, dehydrate, and clear the tissue. The last step of this series is performed in a tissue processing machine, the infiltration of tissues with melted paraffin. The complete processing including fixation takes 18 to 24 hours.  

Embedding

Following the infiltration of heated liquid paraffin, the specimens are ready to be embedded into molds. Paraffin is used to embed tissue because it is capable to convert from liquid to solid form. In addition, paraffin wax supports the enclosed tissue. Therefore, the paraffin block is ready for sectioning.

Sectioning

     
The process of sectioning or cutting in the Histology lab bridges science and art. Experience and training is crucial for microtomy. The paraffin blocks are cut using a rotary microtome. The blocks are chilled in a tray of ice because the cold wax makes a clean cut compared to paraffin wax cut at room temperature. The sections are cut at five microns thick for the microscopic slide. A red blood cell, for example, is eight microns thick which is thicker than the sections cut in our Histology lab. The cut sections form paraffin ribbons and is placed on a hot water bath. The Histotechnologist centers and lifts the paraffin section on the microscopic glass slide. After drying the wet slides, they are ready to stain.  

Staining

Simply put, the nucleus and cytoplasm are the two major components of cells. The purpose of staining is to identify different tissue components through the color reactions.  

Routine Staining

A routine stain is one that stains the various tissue elements with little differentiation except between nucleus and cytoplasm. Hemotoxylin stains the nucleus blue, whereas eosin stains the cytoplasm pink.  

     

Special Staining

Special staining can be used for specific purposes. Special stains are used to determine bacteria, virus, fungi, intercellular, and intracellular structures. Our Histology Department has one special iron stain, which determines if recent or remote hemorrhage is present (subdural hematoma).  

Smears

Smears are taken from the decedent of homicidal cases or one who is a victim of a violent crime. The purpose is to determine if sperm is present.  

Coverslipping

The purpose of a coverslip, or the top glass cover that sandwiches the tissue and glass slide, is both for protection and for microscopic examination of the enclosed tissue.  

The Mounting Reagent

Is a substance that penetrates through the stained tissue, and has a purpose to refract light under the microscope.      

Signing Out

The last step includes entering the case into the computer database and printing the labels. Then we label the slides, and deliver the book of slides to the assigned Pathologist. Note: one book can hold up to twenty slides with one book per case.