FLOATING OUT

• Floating out


  A process in the histopathologic procedures that occurs after sectioning but prior staining. It is done by the histotechnologist and not the histopathologist.
• Floating out is not an easy job. It requires mastery and practice overtime.
• Equipment and materials for floating out

  • Floating-out bath
  • Brush and teasing needle
  • Clean cloth
  • Clean glass slides with frosted ends
• Water


  The solvent used for floating out
• Labelling is very important
• Specimen


  After performing the technological procedures prior staining, we will be able to use a tissue block. Every laboratory has a unique size of the tissue block. We use a paraffinized tissue block for routine laboratory analysis of your specimen.
• Floating out procedure


  1. Pour water in the water bath and turn on the water bath at a temperature approximately 6-10C lower than the melting point of the paraffin wax

  2. Lower one end of the ribbon first and gradually lower the remainder of the ribbon with a slight pull to avoid bubbles

  3. Remove folds and wrinkles by stretching the sections gently with a pair of teasing needles or with a brush

  4. Separate sections by the use of forceps or glass slides

  5. Insert glass slides into the floatation bath perpendicularly

  6. Drain sections approximately for 1 minute
• Tissue sections should not be left in the water bath for a long time. 30 seconds is already enough.
• What happens when floated-out tissue sections are not rapidly transferred in the slide? It will have undue expansion and distortion of the tissue.
• Drying of tissue sections

  1. Place the slide in an oven at 37C to dry overnight

  2. Drying by the paraffin oven, set to 2-5C above the melting point of wax

  3. Drying in incubators set at 37C

  4. Drying at the specified temperature of 45-55C for enzyme digestion, chemical extraction, metallic impregnation, and enzyme localization
• Hot plates are not recommended because they tend to overheat and there is a risk of dust falling onto the sections during the drying period. Presence of dust would not adhere to the slides correctly.
• How to cut sections from tissue embedded in paraffin blocks

  1. Chill paraffin blocks to make sectioning easier

  2. Warm water bath to 40C

  3. Prepare the microtome for sectioning

  4. Insert the blade into the holder

  5. Ensure and secure and set the blade clearance angle and section thickness

  6. Insert the paraffin block and adjust the positioning

  7. Cut a few initial thin sections to check the position of your block and adjust if necessary

  8. Trim the tissue block to expose the tissue surface

  9. Section the tissue at a thickness of about 5 microns

  10. Float the tissue sections on the water bath and allow any wrinkles to disappear

  11. Separate each section in a ribbon by gently tapping then divide with a pair of forceps

  12. Label the slides with the appropriate information

  13. Pick the tissue sections from the water bath, trying to avoid getting any air bubbles underneath

  14. Dry the slides in an oven overnight at 37C
• Rotary Microtome


  • For cutting large blocks of paraffin embedded sections
  • Invented by Minot in 1885-1886 to cut paraffin embedded tissues
  • The most common type used for both routine and research laboratories at present
• Sliding Microtome


  • For cutting celloidin embedded sections
  • Developed by Adams in 1789
  • Favored in laboratories where very hard tissue/blocks are usually sectioned
• Freezing Microtome


  • For cutting unembedded frozen sections
  • Invented by Queckett in 1848
  • Used for STAT (urgent biopsies) specimens
  • Tissue must always be enclosed in a cryostat
• Ultrathin Microtome


  • For cutting sections at 0.5 μ for electron microscopy
  • Employs a diamond knife for cutting
  • Specimens are often small, fixed in osmium tetroxide and embedded in plastic
• Section thickness


  • Paraffin Section: 4-6 μ
  • Celloidin Section: 10-15 μ
  • Frozen Section: 10 μ
  • Ultrathin Section: 500-1200 Å / 50-120 μ
• Angles of microtome knife

  • Bevel Angle: 27-32°
  • Clearance Angle: 5-10°/5-15°
  • Wedge Angle: 14-15°
• Types of microtome knives

  • Plane Concave Knife
  • Biconcave Knife
  • Plane Wedge Knife
• Setting the microtome knife

  1. The knife is usually tilted at 0-15° angulation on a microtome to allow a clearance angle between the cutting facet and the tissue block

  2. Sections are removed in ribbons of ten to allow easy location of serial sections

  3. A section is selected for staining and picked up onto a clean slide in a vertical position

  4. Sections may be flattened out by placing them on a slide which has been flooded with 20% alcohol
• Hot plates are not recommended because they can cause overheating and there is a risk of dust falling onto the section during the drying period.
• Honing


  1. Removal of gross nicks

  2. Heel to toe (Edge first)

  3. Purpose: to remove irregularities from the knife

  4. Hones: Belgium Yellow, Arkansas, Fine Carborundum, Plate-glass hone, Machine hone

  5. Lubricants: Mineral Oil, Clove oil, Xylene, Liquid Paraffin, Soapy water
• Stropping


  1. Removal of burr

  2. Toe to heel (Edge last)

  3. Purpose: polish and sharpen the cutting edge