Histology Techniques

Question 1

What is histology

Answer 1

study of the microscopic anatomy of cells and tissues of plants and animals
~sectioned, stained and mounted on a microscope slide
~enhanced through the use of histological stains

Question 2

What is histopathology

Answer 2

the microscopic study of diseased tissue

important tool in anatomical pathology

Question 3

What are the two types of histological samples that can be taken

Answer 3

from a dead animal (necropsy)

from a live one (biopsy)

Question 4

What are the components of the histology lab

Answer 4

Fixation of tissues
Dehydration, clearing and embedding
Section cutting
Paraffin
Cryostat
Staining sections and mounting coverslips
Photomicrography

Question 5

What is the goal of histological techniques

Answer 5

evenly sectioned (no thick and thin areas),
nicely stained (not over or under stained) with the appropriate stain
no artifacts (rips, tears, air bubbles, bits of dirt or crystals of stain)
in a condition to last many years (properly fixed, coverslip properly mounted)

Question 6

Describe fixation of the tissues

Answer 6

To fix the physical state of the cells, as well as the chemical state.

Allows for the subsequent treatment of the tissue with minimal damage and alteration of the tissue.

Must not interfere with cell components that are active in staining (or they won’t stain)

Question 7

What are characteristics of a well fixed slide

Answer 7

good nuclear and cytoplasmic morphology
minimal shrinkage
clearly defined basement membranes and cell margins.

Question 8

What are the characteristics of a badly fixed slide

Answer 8

Inferior nuclear and cytoplasmic morphology

excessive shrinkage and poorly defined cell margins

Question 9

What is very important for the technician for fixation of cells

Answer 9

Kills microorganisms
~prevents tissue deterioration
~protects the technician handling the tissue from pathogens which might be present.

Question 10

What are examples of chemical fixatives

Answer 10

Formalin: most common
Safe-fix: less toxic
Other:
Special for electron microscopy
Cryostat

Question 11

During fixation can tissue deteriorate?

Answer 11

Yes.
Tissue can deteriorate very rapidly
E.g. Bone marrow
Metabolically active deteriorate fastest
Kidney, liver, pancreas
Should be removed first

Question 12

What is an alternative form of tissue fixation

Answer 12

An alternative method of tissue preservation is perfusion of the animal (often used in research).
Removal of blood from organs by perfusing with physiological saline
Immediate entry of the fixative (normally 10% formalin) into all the blood vessels

Question 13

What are the necessary components of dehydration, clearing and embedding

Answer 13

Gets the tissue ready for sectioning (except for sectioning in a cryostat)
We are embedding the tissues in wax
Must remove water from the tissues (replacing with a fluid soluble in both water and wax) (dehydration and clearing).
Cannot be done all at once (would damage the tissue), so series of steps.
Each step is at least one hour, so the process is automated.

Question 14

How do you dehydrate tissue sample

Answer 14

Remove water
Using alcohol in increasing concentration
Makes the tissue firm for cutting
Prevents shrinkage in paraffin

Question 15

How do you clean the tissue sample

Answer 15

Replaces the alcohol with a liquid compatible with paraffin
Increases tissue transparency
Toluol and xylene are mostly used

Question 16

How do you embed the tissue sample

Answer 16

Makes tissue firmer to prepare for actual block preparation

This last step needs to be timed so that the tissue can be removed as soon as it is ready

Question 17

Why do we trim the tissues before embedding

Answer 17

smaller pieces should be cut for treatment, so that a cut surface suitable for sectioning is prepared.
This is also a time when excess fatty and connective tissue can be cleaned off the outside of an organ.

Question 18

Describe block embedding

Answer 18

Done at the embedding machine, which has a liquid paraffin dispenser.
set at 56C-60C
A thin layer of paraplast (liquid) is put in plastic embedding mold.
The paraplast impregnated tissue is then added
The paraplast should have cooled slightly, just starting to form a thin skin, when the tissue is placed in it.

Question 19

What is the microtome and what is it used for

Answer 19

machine equipped with a very sharp knife and a mechanism to advance the tissue in very small increments.

Tissues are normally sliced at 6 - 10 microns. We use a rotary microtome, which uses a wheel to advance the tissue block.

The knife should be clean, sharp and cold for best sectioning. Sharpen the knife and store it in the freezer (or overnight) before use.

Question 20

What are the steps for cutting things with a microtome

Answer 20

Fasten block
Adjust for straightness
Bring forward blade so block is almost or just touching.
Cut (advance to tissue) (10-15 microns) until a whole section is being taken.
When ready to take good sections, be sure the cutting part of the knife is sharp (reposition it if necessary).
Reduce thickness
Turn the wheel slowly and smoothly to get even sections (hard tissues can be cut more quickly).
The sections should come off in a ribbon.

Question 21

How do you float tissues

Answer 21

The ribbon is placed on paper or in box, and the sections are cut apart and transferred to a floating-out bath which contains a very weak gelatin solution at about 45C .
Alternately a ribbon can be transferred to the bath and then the sections separated.
Ribbons need to be spread out.

Question 22

Describe staining tissues

Answer 22

The goal of a stain or stains is to allow examination of the various characteristics and relationships of the cells.
Different tissues, and different cell components, attract different dyes and stains.
Hematoxylin and eosin, the most commonly used pair of stains, are attracted by different cell components,

Question 23

Describe H/E staining

Answer 23

Slide must be dry
Stains are in solution
For paraffin-impregnated tissue sections, the wax is removed by xylene, then the tissue is rehydrated by moving from 100% to 95% to 80 or 70% alcohol.
The tissue is then placed in water before staining.
After staining the tissue section is dehydrated with 95% and 100% alcohol, and the alcohol is removed by xylene.
The slide is now ready to have its coverslip mounted.

Question 24

what are the 2 main functions of the mounting medium

Answer 24

It has a refractive index close to that of glass so that the tissue and the slide match
It protects the tissue from physical and chemical injury.

Question 25

What are some types of artifacts on a slide

Answer 25

Air bubbles
fold of tissues
knife cracks
hair
debris