Lesson 1

Paraffin technique

It is a technique in slide preparation where tissues are fixed and embedded in wax, making it easier to cut sections from which are then stained

Excision

• It is the initial and most crucial step in slide preparation.

• Samples are collected through biopsy or within 5 mins after death.

• Tissues are cut into small pieces

5 mins

What is the time limit in the collection of samples?

Biopsy

It is another way of collecting sample from a live specimen not from a dead one

2.5 × 2.0 × 0.4 cm

What measurement should the tissue slices should not exceed in a standard tissue processing cassette?

Fixation

It is the 2nd step where tissues are preserved and stops post-mortem autolysis by inactivating autolytic enzymes

10% neutral buffered formalin

What is the most common fixative?

Bouin’s or Karnovsky’s

What fixative is preferred for soft delicate structures like the adrenal gland, brain, eyes?

24 - 72 hrs

How long does it usually take for the fixation process?

Shorter

For softer tissues does it take shorter or longer to fixate?

Optimal concentration

This concentration ensures even fixation throughout the sample, preserving structural details

Too low

This concentration, the tissue may not be fully fixed, risking decay and loss of structural integrity

Too high

This concentration, causes rapid fixation of the outer tissue layers can cause them to become overly dense, preventing formalin from penetrating and fixing the inner layers, leaving them under-fixed and prone to autolysis

Washing

This is the 3rd step where:

• Washing of fixed samples

• Remove excess fixative

• Cassette/gauze is immersed in gently running tap water for 15 to 30 mins.

15 - 30 mins

How long should the fixed sample be immersed in gently running tap water?

Dehydration

This is the 4th step:

• To remove 75% of water in tissues

• Water is not miscible with paraffin wax

75%

How much water is removed during the dehydration process?

No

Is water miscible with paraffin wax?

Ethanol

What is the dehydrating agent used in increasing concentration

Preventing shrinkage

What is the reason for gradually increasing concentration of dehydrating agent?

15 15 15 15 30 45

What is the sequence of duration in minutes for each cycle of dehydration?

70 90 100 100 100 100

What is the sequence of concentration in percent for each cycle of dehydration?

Clearing

This is the 5th step:

• To remove alcohol introduced during dehydration

Xylene

What is the clearing agent used?

No

Is alcohol miscible with paraffin?

20 20 45

What is the sequence of duration in minutes of clearing

Embedding

This is the 6th step, to support the tissue during the cutting process without altering the morphology of the specimen

Xylene-soft paraffin mixture

What is the infiltration agent used?

30 30 45

What is the sequence of duration in minutes of embedding?

boats

What is the term for mold used in embedding where the samples are placed?

24 hrs

How long are samples allowed to solidify in embedding?

Sectioning

This is the 7th step where the hardened block of tissue and surrounding embedding medium is trimmed and placed for sectioning in an instrument called a microtome

Microtome

What is the device used in sectioning?

Serial sectioning

It is where hundreds of sections can be obtained from a single specimen

3.0 - 6.0 um

What is the size of each section in serial sectioning?

Ribbon of section

What is the term for a series of sections from serial sectioning?

Water bath

Where are the ribbon section placed to help remove wrinkles and spread the specimen?

58 deg C temp

At what temp are the glass slides kept in the slide warmer to ensure adhesion?

15 - 20 min

How long are the glass slides are kept in the slide warmer to ensure adhesion?

Adhesive coated glass slide

After letting the sections float in a water bath where are they then placed?

Staining

This is the 8th step where stains are used to enhance the visualization of cellular components, each will highlight a different cellular structure

20 20 30 115 15 15 15-30

What is the sequence of duration in minutes of staining?

I II 100 95 85 70 water

What is the sequence of chemicals used and concentration in staining?