Processing and Embedding

Three main steps of processing

1. Dehydration

2. Clearing

3. Infiltration

Define Dehydration

the removal of free (unbound) water

Define clearing

removes the dehydrating reagent and makes the tissue receptive to the infiltration medium

Define Infiltration

the infiltrating medium penetrates the tissue to hold the cells and intercellular structures firmly in place while they are being cut

The basis of tissue processing

remove all water from the sample and replace it with a media that will provide support for the tissue structures

Factors that influence Quality (3 main, 5 sub)

-Time

-Tissue size

-Operating conditions:

1. Temperature

2. Vaccuum

3. Pressure

4. Agitation

5. Viscosity

-Reagent quality

How does time affect the quality

How does the size of the tissue affect quality

What are the 5 operating conditions that affect quality and in what ways?

How does reagent quality affect the quality of processing

Clarke and Carnoy are ______ soluble. What would be not use and what would we use as a starting reagent?

Water soluble- we would NOT want to use a water based reagent (70% alcohol)

We would start it at 100% alcohol

What are the two methods of dehydration? Define them. Which one is more commonly used?

hydrophilic (water-loving) properties- 

most common- Using reagents (e.g. ethanol) with strong polar groups, attract water and remove it from the tissue using its hydrophilic or water-loving properties

Repeated dilution- works by exposing the tissue to multiple changes of the same, fresh reagent which over time removes all the water

Why is it important that we remove unbound water from the tissue?

Removal of unbound or free water is necessary because most infiltrating media that provide structural support will not penetrate into tissue that has residual water in it

Why do we NOT remove bound water from the tissue?

Removal of bound water can lead to hard, brittle tissue that is difficult to cut

Dehydration displaces any remaining fixative in the tissue

True

Explain graded reagents and why we use this process

Wha tis prevented when we use graded reagents?

Buffer salts

What are the common dehydrating reagents

• Alcohol

  • Ethanol (most common)

  • Methanol

  • Isopropanol

  • Butanol

• Acetone

Due to their high _________, most clearants will leave the tissue transparent

Refractive index

Common clearing agents 

• Acetone

• Aliphatic hydrocarbons

• Aviation gasoline

• Benzene

• Chloroform

• Limonene reagents

• Toluene

• Xylene

What is infiltration also known as?

Impregnation

Infiltration reagents

• Agar

• Gelatin

• Paraffin

• Plastics (GMA and epoxy resins)

Define Universal solvent

Identify the Universal solvents