Week 6

Question 1

What are the instruments in Microtomy that need temperature control

Answer 1

Tissue processor - 2-4 C above paraffin MP

Embedding center - 2-4 C above paraffin MP

Dry air over - - 2-4 C above paraffin MP

Floatation bath - 5-10 below the MP of the paraffin

Question 2

What does a light or compound microscope have

Answer 2

Two magnifying lenses
-objective attacked to rotating nosepiece
-ocular (eye pieces)

Set Kohler, scan at 10, zoom at 40

Question 3

how to calculate total magnification

Answer 3

multiply magnification of the objective lens
and the ocular lens.

x10 ocular and x45 objective would give a total
magnification of x450

Question 4

What is resolution or resolving power

Answer 4

-gives fine details
-ability to differentiate between objects

The smallest distance where two objects are seen as two and not blurred into one

Question 5

what is the path from eyes to lamp

Answer 5

eyes- eyepiece-objective- specimen-condenser-iris diaphragm-field diaphragm-lamp

Question 6

what is chromatic aberration

Answer 6

white after entering a lens is split into colors of the visible spectrum and refracted at different angles with different focus point.
A lens that has not been corrected will show with colored border
The refractive index decreases with increasing wavelength .

Question 7

what type of microscope corrective objectives are used

Answer 7

Achromatic objective - correct for red and blue

Parfocal objective -all objectives are focused in the same plane so magnification can be changed without needing to refocus but you see may need to adjust oculars for parfocality

Question 8

What is micrometery

Answer 8

-uses a micrometer scale in one eyepiece and stage micrometer

-used for measuring or counting on mm scale

Question 9

how to keep microscope in maintenance

Answer 9

-keep covered
-clean lenses with lens paper
-remove oil immediately
-dont dismantle objectives
-keep light off when not in use
-remove slides off stage once done
-focus up not down especially on 40x

Question 10

what is a polarizing microscope

Answer 10

-used to ID crystals (talc, silica, urates) and amyloid stained with Congo red
-target amyloid on tissue is confirmed with this microscope by seeing the apple green birefringence/double refraction/anisotropism (under a light microscope it will appear salmon)

– Birefringence - transmission of light unequally in
different directions.
– Anisotropism – having unlike properties in different
directions

Question 11

how does the polarizing microscope work

Answer 11

-light from this microscope vibrates on one plane
-two filters : Polarizer between light and specimen
Analyzer between specimen and eye
-The analyzer is rotated so optical paths are perpendicular with polarizer and the field will go dark
-birefringent will appear bright on dark background

Question 12

When is phase contrast microscopy used

Answer 12

-for unstained samples with living cells allowing transparent objects to be seen clearly

Question 13

What is dark field microscopy

Answer 13

-used for fine structures -unstained micro orgs or radioactive labelling with silver
-rare in histopath lab
-NO TRANSMITTED LIGHT only scattered light
-stray light is directed on objects and they will be self luminous on a dark background and they will also look bigger

Question 14

What is fluorescent microscopy

Answer 14

-one wavelength of light is absorbed and remitted as light of longer wavelength
-object is hit with short wavelength rays in UV range and light is emitted is violet or blue in visible range
-uses a halogen or mercury lamp with exciter lens and barrier lens

Question 15

What is fluorescent microscopy used for

Answer 15

Autofluorescence - when substances like collagen fibers fluoresce naturally
-used for AB -AG reaction IHC
-* Acid fast bacilli (tagged with auramine – rhodamine) and amyloid (tagged with thioflavin T) can be identified using this technique when they get tagged by fluorescent dye.

Question 16

What is electron microscopy

Answer 16

-high resolution
-electron gun instead of light source
-electron gun, electron beam and specimen are maintained in a vacuum
-Electron beam is directed at sample and focused by changing the strength of the EMF
-sample image is seen by projection onto a fluorescent screen

Question 17

What is transmission EM

Answer 17

• A thin section either transmits electrons (clear areas) or deflects electrons (dark areas).
• A 2-D image is produced.
• For the diagnosis of muscle and kidney diseases, different type of tumors.

Question 18

What is SCANNING E.M

Answer 18

-Electron beam sweeps the surface of the specimen
and releases secondary electrons.
* Produces a 3-D image.
* Used in research to study the surfaces of the surface membranes of cells.

Question 19

What are different types of microtome
**mostly used in pathology labs

Answer 19

Ultramicrotome
– Used for cutting very thin sections 0.5 µm.

• rotary microtome**
  – Most commonly used for paraffin sectioning and found in most cryostats.
• Sliding microtome**
  – Use for sectioning celloidin and large paraffin blocks.
• Clinical freezing microtome**
  – Replaced by cryostat but easier to obtain free floating sections for special stains.

Question 20

What are the parts of a flotation bath

Answer 20

• Set at 5-10 C < MP of wax
• Remove bubbles.
• can use adhesives on slides (albumin, silane, glue, poly-L-lysine, gelatin, agar).
• Charged slides – electrostatic attachment.
• Clean the water surface regularly.
• Use chemicals to lower the surface tension – help flatten out the tissues.

Question 21

Why are dryers and ovens used in microtomy

Answer 21

-to help dewax the slide before staining and introducing water to prep for staining.
-if there is incomplete drying the tissues can wash off during staining and cause incomplete dewaxation
-must be maintained at 60C for routine and 37 for enzyme, ISH, IHC stains
-if there samples get too hot there will be nuclear bubbling, parched earth and cracking

Question 22

What are robotic stainers like

Answer 22

-flexible because you can program it
-allows EXACT staining time
-can do many types of stains at once
-reduce chemical exposure

Question 23

What are linear stainers

Answer 23

-move slides from one container to next
-good for progressive H&E but less flexible for regressive
-can be continuously loaded

Question 24

What is a coverslipper

Answer 24

-automated coverslippers use film coverslips or glass coverslips
-reduce chemical exposure
-needs regular cleaning
-ensure there is correct xylene to resin ratio for glass coverslip

Question 25

What are slide and cassette printers

Answer 25

-cassette ink resists chemicals through processing and staining
-can track location, status, reduces human error

Question 26

What is the gravimetric factor

Answer 26

-method that compensates for variances in dye content of dry dyes that are used in the preparation of staining solutions

present dye/new dye