This document provides an overview of various instruments and equipment used in a histology laboratory, including light microscopes, polarizing microscopes, fluorescence microscopes, electron microscopes, microtomes, tissue processors, stains, and cover slippers. It describes the basic functions and proper maintenance of each tool to ensure quality samples and results.

1. Instrumentation
York College CUNY
HPMT 332
WEEK 4
SPRING 2014
Professor. Emsley

2. Different Instruments/Equipments
found in the Histology Laboratory
I. Light Microscope
II. Polarizing Microscope
III. Fluorescence Microscope
IV. Electron Microscope
V. Rotary Microtome
VI. Sliding Microtome
VII. Cryostat
VIII. Troubleshooting Microtomy
IX. Automatic Tissue Processor
X. Microwave Processor
XI. Manual Staining
XII. Automatic Stainer
XIII. Floatation Bath

3. Light Microscope
• The role of the Light microscope is to magnify an image to a level at
which the retina can resolve the information that would otherwise be
below its limit of resolution.
• Simplest microscope: magnifying glass (1 Lens) or reading glasses..
• Light microscope used for the examination of tissue sections
combines 2 simple microscopes or magnifying glass lens systems
therefore, the light microscope is called a compound microscope.
• Lens systems consists of the objectives lenses and ocular lenses or
eye pieces.
• When white light enters a lens, it is split (refracted) into the colors of
the visible light spectrum.

4. Maintenance of the Light
Microscope
• Keep microscope covered when not in use.
• Clean the lenses frequently with lens paper. Do not use other paper
tissue.
• Remove Immersion oil immediately after use.
• Use xylene on the objective only as a last resort, but if
necessary,use it sparingly and remove it immediately.
• Do not dismantle the objectives.
• Reduce the light to a minimum or turn it off when not in use.
• Do not touch the surface of the lens.

5. Polarizing Microscope
• Polarizing microscope is used mainly for the identification of crystals
• - example talc, silica or urate. People with gout has urate crystals.
• The crystals split the light rays because of its uneven optical
density, and the rays are refracted or bent to differing degrees. This
property is birefringence ( transmitting light unequally in different
directions)
• A compound microscope / light microscope may be converted easily
to a polarizing by placing 1 piece of polarizing film on top of the light
source ( polarizer) and another on top of the microscopic slide
( analyzer). The polarizer is then rotated.
• Through the polarizing microscope amyloid stained with Congo Red
will exhibit an apple green birefringence ( positive for amyloid).The
disease associated to this phenomenon is amyloidosis, -a disease
characterized by an amorphous, eosinophilic, extracellular deposit
that gradually replaces cellular elements of vital organs and causes
progressive loss of function and eventual death.

6. Fluorescence Microscope
• Fluorescence: an optical phenomenon in which light of one
wavelength is absorbed by a substance and almost instantly re-emitted
as light of a longer wavelength.
• The fluorescence microscope makes use of the ability of certain
molecules to fluorescence under ultraviolet light (UV).
• The fluorescence microscope is used to display naturally occurring
fluorescent (auto fluorescent) molecules such as Vitamin A and
some neurotransmitters.

7. Electron Microscope (EM)
• The EM- obtains extra resolving power by replacing the ordinary
light source of a light microscope with an electron gun ie. An
electrified tungsten filament that emits electrons.
• An electron beam has a much shorter wavelength than visible light.
• 2 types of Ems (Transmission and Scanning)
• TEM – very useful in diagnosis of muscle and kidney disease and
tumor identiftcation.
• SEM- has a great depth of focus.
• - it is used to study the surface of an object or specimen and has
been used to study cell surface membrane changes in the evolution
of malignancy and other pathologic processes. This type of
microscope is used mainly for research.
• Ems are very expensive.

8. Rotary Microtome
• Rotary microtome- manual or computerized motor- semi or fully automatic.
• Used for microtomy ( sectioning)
• During lab session detailed explanation / demonstration
• Maintenance of the microtome – always lock the microtome and remove
blades before attempting to clean all debris.
• Use a soft brush or gauze to clean debris and dispose in biohazard bags.
• Cover microtome when not in use.
• Currently microtomes are designed for ergonomic purpose. Semi-automatic
microtomes have pedals or hand controlled devices.
• The fully automatic microtomes have motors that turn the wheel allowing
both hands to be free to manipulate the ribbons and most have foot pedals
to control starting and stopping the rotations.
• In routine cases the microtome is set at 4 or 5 μ

9. Cryostat
Cryostat: a cryostat is a microtome enclosed in a
refrigerated cabinet.
• Often times, during a surgery rapid diagnosis is necessary
therefore, frozen sections have to be preformed.
• Frozen sections are cut on a cryostat.
• Fresh tissue frozen, using a gel-like substance as support medium.
• The gel can withstand freezing temperatures of -50°C to -60°C.
• Some labs use liquid nitrogen to freeze the gel but its very
expensive and difficult to store in smaller labs. Therefore, 2-
methylbutane (isopentane) is used instead of liquid nitrogen.

10. Maintenance of the Cryostat
• Defrost weekly.
• Use ppe and steel mesh gloves to clean and remove knife blade.
• Remove debris from instrument.
• Use slightly damp cloth with 70% alcohol to clean.
• If microbial agent is suspected use 5% amphyl or 10% bleach to
clean out instrument.
• Wipe dry then instrument back on.

11. Sliding Microtome
• The block is held stationary on the sliding microtome , and the knife
is moved along a horizontal plate past the block face.
• This type of microtome is used for sectioning celloidin and large
paraffin blocks.
• It is not used in routine histopathology laboratories.

12. Troubleshooting Microtomy
Microtomy Problems
• Most microtomy problems such as irregular, skipped, thick or thin
sections are usually the result of either too little (fig. 3.3a) or too
much ( fig. 3.3b) blade tilt ( clearance angle) can be corrected by
adjusting the blade so that the clearance angle between blade and
specimen is correct. Grooved, deformed sections are produced by
dull edge , correct by moving to a new section of blade.
• Regular lengthwise or vertical scratches and splits in the sections
are caused by nicks, calcium, bone, or other hard materials and can
be corrected by surface decal of block, or use new blade.
• Mushy sections- insufficient dehydration. Correct by re-processing
the specimen.

13. Automatic Tissue Processor
• Conventional Tissue Processor: processes specimen overnight for
about 16-18 hours. It is operated in a closed system which assist
with keeping exposure to toxic vapors to a minimum when they are
vented to the outside air or through a filter.
• A major advantage of the closed processor is that specimens
cannot dry out in the retort chamber in the event of a malfunction.
• Several reagents are placed on the processor 10% neutral buffered
formalin, 70% alcohol, 80% alcohol, 95% alcohol, 100% alcohol,
xylene and paraffin.
• Automatic tissue processors utilize heat at approximately 45°C
temperature along with vacuum and pressure to aid in the
processing of the tissues.
• Precaution should be taken with increased temperatures on small
biopsies.

14. Maintenance of Tissue Processor
• Clean outside of instrument with xylene dampened cloth.
• Close retort chamber, press CLEAN for the clean cycle to start
automatically
• A Quality Control chart is recommended to ensure that reagents
are monitored and changed frequently as per manufacturer’s
instruction.

15. Manual Staining
• Manually deparaffinize and hydrate slides.
• Hematoxylin ( nuclear Stain)
• Bluing Reagent
• Acid alcohol (differentiation)
• Eosin ( counter stain , cytoplasmic stain)
• Hydrating alcohols
• Dehydrating alcohols

16. Automatic Stainer
• Some Automatic stainers can be programmed to do routine H+E or
Special Stains.
• Manual H+E staining can be time consuming and doesn’t give
consistent results.

17. Maintenance of the Stainer
• Use 10% bleach to clean to clean inside of Stainer.
• Clean hematoxylin containers with 10% bleach and rinse well with
running tap water and test for the presence of chemicals.
• Change reagents when they get dirty or when too many slides were
stained for a day .
• Absolute alcohol containers should be washed then wiped dry to
remove any trace of water.
• Xylene containers should be washed and dried to remove any trace
of water.

18. Manual Cover slipping
• Slides are manually cover slipped to protect the delicate tissues on
the slides.
• AUTOMATIC COVER SLIPPING
• Done automatically by instrument. Cover glass are loaded on this
instrument, with xylene and non- aqueous mounting media.

19. Flotation Bath
• Flotation baths are used for floating out paraffin ribbons.
• The temperature of the bath is usually maintained 5°C to 10°C
below the melting point of the paraffin used for embedding.
• Ribbons should be stretched gently as they are placed on the
flotation bath.
• Cold water in water baths will cause the sections to shrivel up.
• Very warm or hot water will cause sections to disintegrate rapidly.

20. Maintenance of the Flotation Bath
• Remove used water at the end of the day.
• Clean bath with soap and water.
• Place fresh distilled water in water bath .

21. Review
• Explain the advantages and disadvantages of frozen
section versus permanent sections.
• You have just cut a ribbon containing 5 paraffin sections.
As the sections touch the surface of the water in the
floatation bath they disintegrated. What caused the
section to disintegrate?
• The hematoxylin container should be cleaned with 10%
bleach solution and washed thoroughly. The container
should be tested for chemicals before pouring fresh
hematoxylin. Why should the containers be tested for
chemicals?