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How we make slides

Pathologists study patient slides carefully to make their assessments with the aide of sophisticated microscopes. Histology is the science dealing with the microscopic identification of cells and tissue. Before the pathologist can begin this process, a slide has to be made. The following guide will take you through the steps required to produce a slide.

Click to thumbnails to enlarge.

Fixation
Tissues must be immersed in fixative immediately after removal from the body to prevent decomposition, autolysis and artifact formation. 10% Neutral Buffered Formalin is the routine fixative.

1. Specimen Accessioning
Upon verification that the specimen was received in the proper fixative, the specimen is then assigned an accession number. This accession number consists of a letter-number code used to identify the specimen throughout the complete process.

2. Gross Specimen Examination
A pathologist, pathologist assistant or resident visually examines the specimen. A description of the specimen is recorded and the specimen is then cut into the proper size and placed into a color-coded cassette labeled with the assigned alpha-numeric code.

  Inking a gross specimen for margins (not shown) Different color tattoo inks are used to identify margins or other structures of tissue.


3. Tissue Processing
After the specimens are macroscopically examined and placed into the color-coded cassettes, the cassettes are then loaded into baskets that are placed into a tissue processor. Tissue processing consists of four steps: Fixation, Dehydration, Clearing and Infiltration lasting between 8 to 12 hours.

Tissue Processor
A tissue processor is a machine that automatically transfers one chemical to the next inside the cassette holding chamber.
Tissue Embedding
Properly oriented and arranged tissues are cast into liquified paraffin that hardens upon cooling. Enclosing the tissue in the infiltration medium (paraffin) used for processing and then allowing the medium to solidify.
Sectioning with a microtome
The microtome advances the tissue block, toward a sharp knife. The tissue is moved in an up and down motion to cut the tissue into a preset number of micrometers. The paraffin and tissue shavings are shaved off like a ribbon.
The ribbon of tissue is then carefully placed into a water bath. The water bath is a dish of heated, distilled water. The paraffin ribbon floats on top of the water.
Unstained section on glass slide
Placing a positively charged slide under the section and lifting up picks up the tissue section. The tissue adheres to the charged slide
5. Drying Oven
The unstained slides are then placed into a metal tray that gets placed into a drying oven. The oven is warm and helps the section of tissue adhere to the slide.
6. Tissue Staining
When the slides are removed from the oven, they are placed into an automated staining machine. The slides with the tissue on them are immersed in chemicals and dyes that stain the cells. By applying the colored dyes to the cells, the pathologists are able to view the structure of the tissue better.
  H & E Staining
Hematoxylin and Eosin staining is the routine stain for all tissues. Hematoxylin stains the nucleus and the Eosin stains the cytoplasm of tissue cells.
7) Coverslipping

A protective glass coverslip is attached to the slide with mounting medium. This protects the tissue from being scratched. Better microscopic examination at various magnifications is also obtained by the use of coverslips.

  *Frozen Sections (not shown)
When rapid diagnosis is required, the pathologist may need to perform a frozen section. A frozen section is cut using a cryostat. A cryostat is a refrigerated unit that has a microtome. The tissue is frozen solid enough to be sectioned by the microtome and put directly onto a slide for H & E staining. Frozen sections are stained by hand.
   


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