Histology Techniques Flashcards
What is histology
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
What is histopathology
the microscopic study of diseased tissue
important tool in anatomical pathology
What are the two types of histological samples that can be taken
from a dead animal (necropsy)
from a live one (biopsy)
What are the components of the histology lab
Fixation of tissues Dehydration, clearing and embedding Section cutting Paraffin Cryostat Staining sections and mounting coverslips Photomicrography
What is the goal of histological techniques
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)
Describe fixation of the tissues
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)
What are characteristics of a well fixed slide
good nuclear and cytoplasmic morphology
minimal shrinkage
clearly defined basement membranes and cell margins.
What are the characteristics of a badly fixed slide
Inferior nuclear and cytoplasmic morphology
excessive shrinkage and poorly defined cell margins
What is very important for the technician for fixation of cells
Kills microorganisms
~prevents tissue deterioration
~protects the technician handling the tissue from pathogens which might be present.
What are examples of chemical fixatives
Formalin: most common Safe-fix: less toxic Other: Special for electron microscopy Cryostat
During fixation can tissue deteriorate?
Yes. Tissue can deteriorate very rapidly E.g. Bone marrow Metabolically active deteriorate fastest Kidney, liver, pancreas Should be removed first
What is an alternative form of tissue fixation
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
What are the necessary components of dehydration, clearing and embedding
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.
How do you dehydrate tissue sample
Remove water
Using alcohol in increasing concentration
Makes the tissue firm for cutting
Prevents shrinkage in paraffin
How do you clean the tissue sample
Replaces the alcohol with a liquid compatible with paraffin
Increases tissue transparency
Toluol and xylene are mostly used
How do you embed the tissue sample
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
Why do we trim the tissues before embedding
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.
Describe block embedding
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.
What is the microtome and what is it used for
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.
What are the steps for cutting things with a microtome
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.
How do you float tissues
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.
Describe staining tissues
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,
Describe H/E staining
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.
what are the 2 main functions of the mounting medium
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.
What are some types of artifacts on a slide
Air bubbles fold of tissues knife cracks hair debris
