Histopathology

1. MICROTOME
Mr.Mahmoud Ibrahim

2. Introduction
 At the beginning of light microscopy
sections were manually prepared using
razor blades.
 1st devices invented - George Adams, Jr.
1770
 Developed by Alexander Cummings

3.  The device was hand operated with the
sample held in a cylinder

5.  Andrew Prichard - table based model in 1835
 Allowed for reduction in vibration
 Present day microtome :
 Wilhelm His, Sr. (1865)
 Jan Evangelista Purkyně

6. Microtome
 Machines that cut extremely thin sections
from a sample for applications in histology
or pathology
 Use special metal, glass, or diamond blades,
depending on the type of specimen and the
desired thickness

7.  All microtomes consist of main parts
 Base (microtome body) -
 Knife attachment (Knife holder)

8.  Material or tissue holder
 Advancing mechanism, and a
mechanism for adjusting section
thickness.

9.  Modern microtome are precision
instruments designed to cut uniformly
thin sections
 For electron microscopy, where
magnifications of several hundred
thousands are possible, the thickness of a
section is usually of the order of 10
Nano meters (ultra-thin section)

10. Types of microtomes
 1. Cambridge rocking microtome
 2. Rotary microtome
 3. Base sledge microtome
 4. Sliding microtome
 5. Freezing microtome (cryostat)

11. ROCKING MICROTOME( Cambridge)
 Produced in large numbers early in the 20th
century
 Designed only for cutting paraffin sections
 Oldest in degisn, cheap , simple to use.
 Extremely reliable.

12.  the tissue moves through an arc as it
advances towards the knife (the slightly
biconcave knife is used) causing the
sections to be cut in a curved plane.

13.  Very thin sections are difficult to obtain
and
 one major disadvantage is a limit to the
size of block which can be cut.

14.  Because of the lightness of the frame the
microtome has a tendency to move
during cutting. The rocking microtome
has largely been replaced by the more
precise rotary microtome

15. Cambridge Rocking Microtome

16. ROTARY MICROTOME
 The microtome operation is based upon
the rotary action of a hand wheel
activating the advancement of a block
towards a rigidly held knife.

17.  The block moves up and down in a
vertical plane in relation to the knife and
therefore cuts flat sections.

18.  Available machines range from
lightweight (rotary microtome suitable
for cutting paraffin wax embedded
material in a continuous ribbon),

19.  to heavy duty, motor driven instruments
used to section hard material embedded
in synthetic resin. The rotary microtome
can also be found in most cryostats for
cutting frozen sections.

21. Rotary Microtome

24. Base sledge microtome
 These are designed for cutting large
blocks of paraffin and resin embedded
material including whole organs, for
light microscopy. a major advantage
when sectioning large, hard blocks.

25.  can also be used to cut materials from
various industrial applications (wood,
plastics, textile fibers).

26.  Mechanism of action:
The block holder is mounted on a steel
carriage which slides backwards and
forwards on guides against a fixed
horizontal knife.

27.  Advantages:
 Heavy , very stable, not subject to
vibration
 Paraffin wax embedded sections are
more easily cut .

28.  Disadvantages
 With practice, sections from routine
paraffin blocks can be cut as quickly as
on any other type of microtome.

30. Sliding microtome
 The fundamental difference between the
sliding microtome and those models
described earlier is that with this instrument
the block remains stationary while the
microtome knife moves during the process of
the sectioning .

31.  Designed for cutting celloidin-embedded
tissue blocks.
 The knife or blade is stationary, specimen
slides under it during sectioning.
 Also used for paraffin –wax embedded
sections

34. FREEZING MICROTOME
 This form of microtome is used for cutting thin
to semi-thin sections of fresh, frozen tissue
 The freezing microtome is equipped with a stage
upon which tissue can be quickly frozen using
either liquid carbon dioxide, from a cylinder

35.  Some cooling systems also allow the knife to
be cooled at the same time.
 thin sections are very difficult to obtain with
this type of microtome.

37. ULTRAMICROTOME
 The ultramicrotome is used to prepare
ultrathin sections for light and electron
microscopy.

39. CRYOSTAT
 A cryostat is primarily used for cutting
sections of frozen tissue
 The cryostat commonly consists of a
microtome contained within a refrigerated
chamber, the temperature of which can be
maintained at a preset level.

40.  The cryostat usually contains a rotary
microtome although some portable units
utilize a rocking microtome.

42. Microtome knives
 STEEL KNIVES
Steel microtome knives are manufactured
from high quality steel which is heat treated
to harden edge. The steel should be free
from impurities, contain anti-corrosives..

43.  NON-CORROSIVE KNIVES FOR CRYOSTATS
These are manufactured from hardened,
heat treated stainless steel free from all
impurities and containing 12 to 15%
chromium.

44.  DISPOSABLE BLADES
 TUNGSTEN CARBIDE
 GLASS KNIVES
 DIAMOND KNIVES

45.  Glass microtome blades
 used in sample preparation activities for
light microscopy and electron
microscopy applications

46.  Diamond blades
 may be industrial-grade or gem-quality.
 Industrial-grade blades are used to slice hard
materials such as bone and teeth.
 Gem-quality blades for microtomes are used
mainly in electron microscopy applications

47. Profile of steel knives
 Microtome knives of hardened steel are
made to four different profiles for
cutting various materials

48. Strongly plano concave/biconcave
 One surface of the Plano concave knife
is straight whilst the other is hollow
ground.
 bi-concave knife has two hollow ground
surfaces.

49.  Both knives are extremely sharp and are
used for cutting soft, celloidin embedded
material. These knives are not suitable
for relatively hard materials

52.  wedge Shaped
The wedge shaped knife has more rigidity
than profile A or B knives and can therefore
be used for cutting harder materials.
Because of the extra thick nature of the
wedge at the tip.

53.  More stable
 Most widely used
 Moderately hard samples
 Used for frozen & paraffin section

55. Sharpening of the knifes
 Manual procedure or automatic
procedure.
 1) Abrasive grinding of the facets
[HONING]
 2)Polishing [STROPPING]

56. Abrasive grinding of the facets
[HONING]
 Naturally occurring slabs of stone with
varying abrasive properties:
 Stones : Belgian black vein and Arkansas,
Aloxite and carborundum-composites.
 Lubricated with soapy water or light oil
during use.

57. Abrasives
 Aluminium oxide(alumina)
 Iron oxide(Jeweller’s rouge)
 Silicon carbide
 Diamond

58. KNIFE SHARPENING
 Manual
Coarse honing can be performed manually
using a lapping stick coated with diamond
paste containing industrial diamonds with
sizes up to 25 µm diameter.

59. FINE KNIFE SHARPENING
 7 Fine honing is achieved by applying
diamond paste, containing industrial
diamonds of 1 µm or less, to a lapping
stick which is then moved against the
knife edge. Or by using stones.

60. STROPPING
 Stropping polishes the knife edge and
removes fine metal retained along the edge
after honing. Leather, coated with a fine
powder or a lapping stick coated with a
diamond paste containing industrial
diamonds of less than 0.5 µm

61.  are effective for this purpose. It is
important that very little pressure is
used when stropping.

63. Microtomy- paraffin wax
 Factors involved in producing good
paraffin-wax sections :
 Temperature:
 Tissues are more easily sectioned at a lower
temperature than that of the atmosphere.

64. Knife angle
 Greater the rake angle(flatter the knife)more
likely is a smooth plastic flow type cutting
action.
 Higher rake angles are more suitable to softer
tissues
 Lower rake angles for harder tissues

65. Angles asociated with the knife edge.
A:rake angle; b:bevel ; c:clearance angle.

66. Speed of cutting
 Soft tissues are cut more easily at a slow
speed.
 Hard tissues are cut easily at a little fast
rate

67. Slant
 Commonly used to refer to the
relationship of the knife edge to the
block when cutting nitrocellulose-
embedded tissue on a sliding microtome.

68. Paraffin section cutting
 Equipment required:
 Microtome.
 Flotation(water bath)
 Slide drying oven or hot plate
 Fine pointed or curved forceps.
 Sable or camel haired brush.
 Scalpel.

69.  Slide rack.
 Clean slides.
 Teasing needle.
 Ice tray.
 Chemical resistant pencil or pen.

70. Section adhesives
 Section may be floating off slides during staining
,there are occasions when:
 Sections are submitted to strong alkali solutions
during staining.
 Cyrostat section for immunofluorescence,
immuno cytochemistry, and urgent diagnosis.

71.  Tissue from CNS.
 Sections are submitted to high temperatures.
 Tissues containing blood clot.
 Tissues which have been decalcified.

72.  Examples of adhesives:
 1- Natural adhesives:
 Albumin , Gelatin, Starch,
 Limitation !!
 - Bacterial over growth.
 -Background staining.

73.  2- Recommended synthetic adhesives:
 - Mayer‫י‬s adhesive.
 - poly -L- lysine.
 - Amino Propyl triEthoxy Sialine (APES)