Histological Methods

Question 1

define plane of section

Answer 1

3D structures may appear different when thin sectioned

Question 2

describe types of sections

Answer 2

cross section (circle)
oblique (oblong)
longitudinal (long rectangle)

Question 3

what type of section are most samples in

Answer 3

oblique section

Question 4

why can’t you created a perfect sample (plane of section)

Answer 4

cannot orient sample when in wax
can’t move it after so what is seen is based on plane of section

Question 5

what is the thickness of a section

Answer 5

5um

Question 6

what is the diameter of a cell

Answer 6

10 um

Question 7

what is the diameter of a lysosome

Answer 7

200nm

Question 8

what is the thickness of PM

Answer 8

75A (7.5 nm)

Question 9

describe H&E stain

Answer 9

hematoxylin and eosin

Question 10

describe hematoxylin

Answer 10

basic
stains basophilic structures
blue or purple

Question 11

name structures stained by hematoxylin

Answer 11

cell nucleus and other structures
basophilic

Question 12

describe eosin

Answer 12

acidic
stains eosinophilic structures
pink

Question 13

name structures stained by eosin

Answer 13

cytoplasm and collagen

Question 14

what does basophilic mean (give ex of structures)

Answer 14

structures with nucleic acids
ribosomes
chromatin rich nucleus
cytoplasmic regions rich in RNA

Question 15

what does eosinophilic mean (give ex of structures)

Answer 15

structures composed of intra/extra cellular protein
most of cytoplasm

Question 16

T or F: structures have to be basic/acidic to be dyed a certain colour

Answer 16

FALSE
it’s based on affinity to dyes

Question 17

describe electron microscope (how image is created)

Answer 17

Electron generated by cathode travels in high vacuum and hits photograph plate (phosphorescent screen)

Question 18

what is an electron lucent structure

Answer 18

when electron passes through - so doesn’t hit anything

Question 19

what is an electron dense structure

Answer 19

when electron hits something and is deflected away

Question 20

describe resolving power of EM

Answer 20

2 dots come together
means that em can magnify way more than LM
LM = 1000x
EM = 160000x or 1.6 mil with cam zoom

Question 21

what are the types of ways to look at specimens

Answer 21

light microscopy
electron microscopy

Question 22

name the pathway to LM

Answer 22

chemical fixation
dehydration
clearing
infiltration
embedding
sectioning
mounting
removal of paragon
rehydration
staining
LM
OR
start with freezing —> sectioning and same ones down

Question 23

describe freezing & down for LM

Answer 23

a way to preserve the specimen
then sectioning = done with cryostat

Question 24

describe chemical fixation for LM

Answer 24

formalin
animal is anesthetized

Question 25

what is an advantage of bouins fluid

Answer 25

better for H&E stain

Question 26

what is perfusion and immersion

Answer 26

perfusion = inject into animal and works way inside out immersion = take organ out and drop into fix and works way into tissue want to stop decay and conserve ultrastructure

Question 27

which method is faster: perfusion or immersion

Answer 27

perfusion is better penetrates better and faster

Question 28

describe dehydration for LM

Answer 28

dehydrate to put sample into blocks need to get rid of water so increase concentration of ethanol (and decrease concentration of water) want to use xylene to put tissue into wax blocks but water and xylene don’t mix

Question 29

describe clearing LM

Answer 29

xylene is a paraffin solvent

Question 30

describe infiltration LM

Answer 30

xylene/paraffin adding dilute wax first (easier than adding to straight up wax)

Question 31

describe embedding LM

Answer 31

putting in paraffin

Question 32

describe sectioning LM

Answer 32

microtome like a meat slicer

Question 33

describe mounting LM

Answer 33

using a glass slide

Question 34

describe removal of paraffin LM

Answer 34

using xylene to remove wax

Question 35

describe rehydration LM

Answer 35

describe concentration of ethanol (increase water) so it becomes an AQ solution

Question 36

describe staining LM

Answer 36

H&E and/or histochemical reaction hematoxylin and eosin are water based so must first remove wax (removal of paraffin and rehydration)

Question 37

describe chemical fixation EM

Answer 37

gluteraldehyde and osmium tetroxide = very fast fixative preferably perfusion

Question 38

describe dehydration EM

Answer 38

propylene glycol doesn’t mix with water so must remove water

Question 39

describe clearing EM

Answer 39

propylene glycol epoxy solvent

Question 40

describe infiltration EM

Answer 40

adding dilute plastic propylene glycol/plastic

Question 41

describe embedding EM

Answer 41

straight plastic

Question 42

describe sectioning EM

Answer 42

ultramicrotome very thin slices

Question 43

describe mounting EM

Answer 43

copper grid

Question 44

describe rehydration EM

Answer 44

there is NO REHYDRATION plastic is fine to put on grid

Question 45

describe staining EM

Answer 45

lead citrate and/or histochemical reaction

Question 46

how does EM work

Answer 46

super thin slices go on grid and are blasted with electrons

Question 47

describe what happens when electrons fly at grid/sample

Answer 47

electrons can go through and burn a whole = electron lucent structure electrons can hit lead or grid and it takes off = electron dense structure