microscopes

what two features should a sample have

• contrast

• translucent

9 stages of light microscope tissue processing

• fixation

• decalcination

• dehydration

• clearing

• impregnation

• embedding

• sectioning

• staining

• mounting of coverslip

purpose of fixation

• damage pathogenic microorganisms

• prevent autolysis and putrefaction

most common solution for fixation and why its used

formaline

• maintain structure, increase rate of diffusion

thickness of fixation tissue

2-3mm

when is frozen section technique used

fast preparation of non fixed tissues

purpose of decalcification

make thin and soft sections of sample after minerals have been removed

how is dehydration occuring

removal of water by increasing concentrations of alcohol

how does clearing work

alcohol present in tissues must be removed by an agent in which paraffin and alcohol are soluble in

what does clearing agent increase

refractive index of tissues so makes it more transparent

types of clearing agents

xylene, benzene, cedarwood oil, chloroform, toulene

what happens during process of impregnation

tissue is dissolved in paraffin, cool sample, cut into thin sections by microtome

sectioning

translucent micro sections are made

name

oblique

cross section

longitudinal

rotary microtome + requires light microscope

sledge microtome

cyromicrotomes (frozen in liquid nitrogen chamber)

laser

Saw (hard and brittle)

Ultra microtome

Purpose of staining

highlight structural part of tissues

basic dyes stain what

acidic structures that are negatively charged eg, dna and rna

acidic dyes stain what

basic structures such as positively charged amino acids

what does this show (staining)

reticular fibres stained with silver impregnation

amyloid stained with congo red

PAS stains glycogen in hepatocytes

hematoxylin

basic dye

blue/purple

nucleus, ribosome, rer

eosin

acidic dye

red/pink

cytoplasm, fibres of extracellular matrix

mounting of coverslip done to

protect from external environment and mechanical damage