Laboratory Equipment

Darkfield
A. Microscope
B. Automatic Processors Dark-field microscopy is ideally used to illuminate
C. Microtome unstained samples causing them to appear brightly
D. Microtome Knives lit against a dark background. This type of
E. Hone and Strop microscope contains a special condenser that
F. Tissue Cassettes scatters light and causes it to reflect off the specimen
G. Embedding Molds at an angle.
H. Oven
I. Incubators
J. Tissue Floatation Bath
K. Slide Dryers
L. Staining Jars
M. Slides and Coverslips

Microscope

 Light or Brightfield Microscope

 Dark-field Microscope
 Electron Microscope
 Phase-contrast Microscope
 Polarizing Microscope
 Fluorescent Microscope

Light/Brightfield

With the bright-field microscope, stained tissue is

examined with ordinary light passing through the
preparation
Phase-contrast
Unstained cells and tissue sections, which are usually
transparent and colorless, can be studied with these
modified light microscopes. Cellular detail is
normally difficult to see in unstained tissues because
all parts of the specimen have roughly similar optical
densities. Phase-contrast microscopy, however, uses
a lens system that produces visible images from
transparent objects and, importantly, can be used
with living, cultured cells
Phase-contrast microscopy is based on the principle
that light changes its speed when passing through
cellular and extracellular structures with different
refractive indices.
Polarizing
Polarizing microscopy allows the recognition of
stained or unstained structures made of highly
organized subunits. When normal light passes
through a polarizing filter, it exits vibrating in only
one direction. If a second filter is placed in the
microscope above the first one, with its main axis
perpendicular to the first filter, no light passes
through. If, however, tissue structures containing
oriented macromolecules are located between the
two polarizing filters, their repetitive structurerotates
the axis of the light emerging from the
polarizer and they appear as bright structures
against a dark background The ability to rotate the
direction of vibration of polarized light is called
birefringence and is a feature of crystalline
substances or substances containing highly oriented
molecules, such as cellulose, collagen, microtubules,
and actin filaments.
Electron Microscope
a. Transmission Electron Microscope
b. Scanning Electron Microscope
Transmission and scanning electron microscopes are
based on the interaction of tissue components with
beams of electrons. The wavelength in an electron
beam is much shorter than that of light, allowing a
1000-fold increase in resolution.
Transmission Electron Microscope
The transmission electron microscope (TEM) is an
imaging system that permits resolution around 3 nm.
This high resolution allows isolated particles
magnified as much as 400,000 times to be viewed in
detail. Very thin (40-90 nm), resin-embedded tissue
sections are typically studied by TEM at
magnifications up to approximately 120,000 times
Specimens are scanned through by beams and it
produces 2-dimensional images.
Scanning Electron Microscopy
Scanning electron microscopy (SEM) provides a
high-resolution view of the surfaces of cells, tissues,
and organs. Like the TEM, this microscope produces
and focuses a very narrow beam of electrons, but in
this instrument the beam does not pass through the
specimen. Instead, the surface of the specimen is first
dried and spray-coated with a very thin layer of
heavy metal (often gold) that reflects electrons in a
beam scanning the specimen. The reflected
electrons are captured by a detector, producing
signals that are processed to produce a black-and-
white image. SEM images are usually easy to
interpret because they present a three-dimensional
view that appears to be illuminated in the same way
that large objects are seen with highlights and
shadows caused by light.
Electrons bounce on the specimen’s surface when
scanning and it produces 3-dimensional images
(topographic).
Microtome Purpose: To cut serial sections of large blocks of
paraffin embedded tissues

Inventor: Paldwell Trefall

Year: 1885

Rotary Microtome

3 Major Parts

- Block holder Purpose: for cutting paraffin embedded sections

- Knife holder and the knife
- Adjustment screws and rachet device Inventor: Minot

Year: 1885-1886

Sliding Microtome

Types of Microtomes

 Rocking Microtome Purpose: Used to cut celloidin embedded sections

 Rotary Microtome Inventor: Adams
 Sliding Microtome
 Freezing Microtome Year: 1789
 Ultrathin Microtome Two Types:

A. Standard microtome
Rocking Microtome B. Base ledge microtome
Freezing Microtome Care of the Microtome

- After sectioning, all the accumulated paraffin and

small pieces of tissues must be brushed away
with a soft brush and not allowed to stay in the
microtome.
- After carefully drying the machine and knife
holder, the parts should be wiped with xylol.
- The microtome must always be covered when
not in use, to prevent accumulation of dust and
Purpose: to cut unembedded frozen sections other dirt which may later on interfere with the
normal sectioning of tissues.
Inventor: Queckett

Year: 1848
Automatic Processors
Ultrathin Microtome

Automated tissue processors are of two main types:

tissue-transfer machines and fluid-transfermachines.
The tissue processor finds applications in
Purpose: for electron microscopy
histopathology laboratories to automaticallyprepare
Inventor: tissue samples for laboratory testing, by fixing,
dehydrating, clearing, and infiltrating them with
Year:
paraffin.

One example of an automatic processor also is the

Elliot bench-type processor

Microtome Knives

Biconcave: paraffin, paraplast, and carbowax

embedded blocks = rocking and rotary

Plane concave: celloidin embedded blocks = sliding

Plane wedge: frozen blocks = freezing microtome

 Types

Other Types of Knives 1. Belgium yellow – for manual sharpening when

cutting edge has been rendered blunt or nicked. This
type usually gives the best result.

2. Arkansas – give more polishing effect than the

Belgian yellow.

3. Fine carborundum – is much coarser than the

first two types and is used only for badly nicked
knives followed by either one of the first two
sharpeners.

 Disposable blades Order of sharpening really blunt knives

 Glass knives - Glass knives are used in Fine carborundum -> Belgium yellow -> Arkansas
ultramicrotomy for trimming specimen
blocks and cutting thin and ultrathin sections
 Diamond knives – better than glass knives Strop

Hone

- A leather strip that is used to remove the burrs

formed when honing.
- It is typically done after honing to remove the
A stone that is used to remove the nicks from the burr.
knives

Tissue Cassette

Holds tissue specimens during processing and

embedding, as well as in storage
Embedding Molds
- It is used to mold your tissue block for easier
handling during the cutting
- After fixing and clearing of your tissues, they are
oriented in these embedding molds.
Paraffin Oven
- For infiltration of tissues in your paraffin,
paraplast, carbowax, plastic, and resin
- The temperature is set at a recommended
amount depending on the medium used
- It is also used for drying glass slides and heating
solutions or stains. It hastens staining by making
the specimen absorb it faster.
- Temperature must not exceed 80 degrees
Celsius
Incubator
Some sections require careful drying; thus, they are
put in an incubator.
Tissue Floatation Bath
- Thermostatically controls the temperature
around 6 degrees to 10 degrees Celsius below
that of paraffin’s melting point
- Tissue flotation bath is required after the step of
cutting paraffin sections and before these are
placed on slides. It lets tissues relax and
smoothen before being mounted on slide as well
as makes paraffin stick to the slide. This ensures
removal of wrinkles and folds before sections are
placed on the slide.
Staining Jars
- Staining jars are used to stain specimens such as
bacteria, cells and tissues on microscope slides.
Without proper staining, most specimens are just
a clear substance not easy to observe and study.
- Internal ridges prevent adhesion
Slide Dryer
The dryer is designed to rapidly dry slides by blowing
warm air over the surface of the slides. The slide
dryer accommodates two slide racks directly from a
stainer and allows the slides to be stained in
approximately 20-30 minutes.

Slides and Coverslips

A prepared slide that is made up of a microscope

slide, specimen and a cover slip not only gives the
viewer better control over the specimen, but protects
the microscope as well. The cover slip protects the
ocular lens from damage by acting as a barrier
between it and the specimen.