Tt instrumemtation

Instrumentation

York College CUNY HPMT 332WEEK 4

SPRING 2014Professor. Emsley

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

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.

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.

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.

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.

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.

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

CryostatCryostat: 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.

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.

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.

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.

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.

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.

Manual Staining

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

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.

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.

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.

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.

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 .

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?