microtomy in histopathology

1. MICROTOM
Y
BY EKTA JAJODIA

2. At the beginning of light microscopy sections
were manually prepared using razor blades.
1st devices invented - George Adams, Jr. 1770
Developed by Alexander Cummings
The device was hand operated with the sample
held in a cylinder

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

4. DEFINITION
Microtomy – means by which tissue can be
sectioned and attached to a surface for further
microscopy
Microtome -
oMikros - "small“
oTemnein - "to cut“
oSectioning instrument for cutting extremely
thin slices of tissues

5. MICROTOME KNIVES

6. Greatest single factor in producing good
sections
It should be :
oSharp
oWell adjusted

7. DESIGN AND SHAPES
Planar concave knives –
oextremely sharp
overy delicate
oused with very soft samples

8. Wedge shaped knives –
oMore stable
oMost widely used
oModerately hard samples
oUsed for frozen & paraffin section

9. Chisel shaped -
oblunt edge
oraises stability of knife
orequire more force to achieve the cut

10. Bi-concave knives – recommended for:
Paraffin section cutting on –
oRocking microtome
oSledge type microtome

11. MATERIALS
USED
 Steel blades - prepare sections for light microscopy
& frozen sections
 Glass blades - for light microscopy and semi-thin
sections for electron microscopy
 Diamond blades - for hard materials like bone, teeth
for both light & electron microscopy
 Disposable blades – for routine microtomy &
cryotomy

12. DISPOSABLE BLADES
Coated with Polytetraflouroethylene
 Mainstay in most laboratories
 Advantages :
o Reliability of constant sharp edge
o Ease to use
o Low & high profiles adaptable to variety of tissue &
paraffin types
o Low cost

13. ANGLES OF MICROTOMY
KNIVES
 Cutting edge (radius of curvature - 0.1 – 0.35 μm) –
is the straight line formed by intersection of 2 planes,
the cutting facets (length – 0.1- 0.6 mm)
 Bevel angle ( 27⁰ - 32⁰) – angle between the planes
Wedge angle ( 15⁰) – angle between the sides of the
knives
 Clearance angle (5⁰-10⁰)- angle between cutting
facet and block of tissue
 Rake angle - angle between upper surface of the
cutting facet and the surface of the block

15. HONING
 Done to restore straight cutting edge & to correct
bevel
 Types of Hone :
o Belgian Black Vein
o Arkansas
o Aloxite
o Tam O’ Shanter Scotch Hone
o Carborundum
o Plate Glass

16. ABRASIVES AND
LUBRICANTS
ABRASIVES LUBRICANTS
 Aluminium oxide
 Iron oxide
 Silicon carbide
 Diamond
 Paraffin oil
 Vegetable oil
 Soap water

17. Belgian Black Vein
 Best & Fast Hone
 1/2″ thick yellow stone backed with ½″ thick
black stone
 Yellow side used for honing
 Used for coarse grinding & finishing

18. PLATE GLASS
 Used by applying an abrasive like aluminium
oxide
 Advantage – Used for honing of all types of
blades by changing the abrasive powder

19. TECHNIQUE OF
HONING

20. KNIFE SHARPENING MACHINE
 For quick honing within 10 mins
 Unskilled junior can also obtain good finish
 Employs revolving cast iron/glass wheel
 Lapping compound like alumina(coarse/fine),
suspension fluid & water used
 Proper cleaning & maintenance of the machine is
necessary
 Fully automatic knife sharpener – employs high
frequency vibrating hone plate.

21. STROPPING
 Process of polishing an already fairly sharp edge
 Blunt knife cannot be sharpened on a strop
 Types :
o Flexible/Hanging
o Rigid
 Best strops are made from hide from the rump of
horse & are marked “shell horse”

22. TECHNIQUE OF STROPPING

23. TYPES OF MICROTOME
Rotary Microtome
Base Sledge Microtome
Rocking Microtome
Sliding Microtome
Freezing Microtome
Ultramicrotomes

24. ROTARY MICROTOME
 Referred to as “MINOT” after its inventor
 Mechanism – rotation of a fine advance hand
wheel through 360⁰ moving the specimen vertically
past the cutting surface & returning to original
position
It may be –
1.Manual (completely manipulated by operator)
2.Semi-automated (one motor)
3.Fully automated (two motors)

25.  Advantages – 1. Ability to cut thin 2-3mm sections
2. Easy adaptation to all types of tissue sectioning
(hard, fragile, fatty)
3. Ideal for cutting serial sections
 Disadvantage – Manual ones can cause repetitive
motion disorder
Advantages of automated rotary microtome –
o Improved section quality
o Increased productivity
o Occupational safety for technologist
o Reduced incidence of repetitive motion disorders

26. ROTARY MICROTOME

28. SLIDING MICROTOME
 Knife/Blade is stationary and specimen slides
under it
 Developed for use with celoidin-embedded
tissue blocks

29. SLIDING MICROTOME

30. BASE SLEDGE MICROTOME
Specimen is held stationary
Knife slides across the top of the specimen
Used for –
oLarge blocks
oHard tissue
oWhole mounts
oNeuropathology & ophthalmic pathology

31. BASE SLEDGE
MICROTOME

33. ROTARY ROCKING MICROTOME
The name of the apparatus comes from the rocking
action of the cross arm
Knife is fixed
Tissue block moves through an arc & strikes against
the knife
Used in early cryostats
Disadvantages –
o Size of blocks that can be cut is limited
o Sections are cut in a curved plane

34. ROTARY ROCKING MICROTOME

35. Freezing microtome:
oUsed for frozen section
Ultramicrotome:
oUsed for electron microscopy

36. Freezing microtome
Ultramicrotome

37. PARAFFIN SECTION
CUTTING
Equipment required –
Floatation (water bath)
Slide drying oven or hot plate
Fine pointed forceps
Camel haired brush
Scalpel, slide rack, clean slides
Teasing needle
Ice tray
Chemical resistant pencil or pen

38. WATER BATH / FLOATATION BATH
A thermostatically controlled water bath used for
floating out tissue ribbons after sectioning
Temperature of water bath - 10°C below the melting
point of the paraffin
To prevent water bubbles trapped under the section
distilled water is used (tap water gives rise to air
bubbles)
Alcohol or a small drop of detergent may be added to
water to reduce the surface tension

39.  2-3 drops of thymol can be added to prevent
bacterial growth - this will allow the water in the bath
to be used over several days

40. DRYING OVEN / HOT PLATE
Temperature should be equal to the melting point of
paraffin
Lower temperature (37°C for 24 hrs ) required for -
oDrying delicate tissues
oTissues from the CNS
Too hot oven may lead to -
oDistortion of cells
o Loss nuclear detail
oDark pyknotic nuclei

41.  Complete drying occurs in 30 mins
 In our automated staining machine – requires only
10 mins ( at 65 degree celcius)

42. BRUSH AND FORCEPS
Used for removal of folds, creases and bubbles
Also used for manipulating the section as it passes
across the edge of the blade

43. SLIDES
 (76*25) mm are universally used
 thickness – 1-1.2mm
 Slides with ground and polished edges should be
used to avoid danger of finger cuts
 Identification details are made on slide by
diamond markers
 Preferred is frost-ended slides – identification
mark inscribed using a soft pencil

44. SECTION ADHESIVES
Used to prevent section detachment from slide
during staining
Causes of section detachment –
oExposure to strong alkali solution during staining
oCryostat section for immunofluorescence,
immunohistochemistry or intraoperative diagnosis
oCNS tissues
oExtreme temperature
oTissue containing blood and mucus
o Decalcified tissues

45. Adhesive commonly used are-o
Mayer’s egg albumin-glycerol
o Dilute serum or plasma
o Gelatin in floating out bath
o Starch
o Poly-L-Lysine
o 3-Amino Propyltriethoxysilane( APES)
o Charged or Plus slides

46. Disadvantage of protein adhesives -
oMay give a dirty background
oProne to bacterial overgrowth
oProne to heavy staining
 2 adhesives are favoured –
1. Poly-L-lysine – we use for IHC, IF and cryostat
2. 3-APES
 For routine HPE – we use Meyer’s egg albumin

47. Practical microtomy

48. TRIMMING OF TISSUE BLOCKS
 An old knife/blade may be used
 Trimming done at 15microm thickness for almost
all tissues
 For small biopsies like endoscopic biopsies and
core biopsies of renal or liver – 10 microm
Aggressive trimming will cause moth-hole artifact

49.  After trimming – block is placed on ice for some
minutes
ADVANTAGES of this – 1. Cooling both tissue and
wax and giving them a similar consistency
2. A small amount of water gets absorbed into the
tissue, slightly swelling it and making sectioning
easier
 Routine sections are cut at 3-4 μm
 Serial sections are best cut in ribbons of 10-12
inches in length, successive section sticking edge-to-edge

50.  In difficulty during sectioning, block face is warmed
by-oWarm
water
oExhaled air
 After cutting ribbons the first section is held by
forceps and the last section eased from the knife
edge

51. FLOATING OUT SEC TIONS
 Sections are floated in the water bath to remove
folds
 Ideal floatation time is 30 seconds
 Prolonged floatation causes tissue expansion &
distortion
 Pre-flooded slides with 50% alcohol – used for
circular structure like eye
 Water bath to be cleaned to remove debris after
each cut
Done by dragging tissue paper across the surface

52. DRYING SECTIONS
 Small amount of water held under the section will
allow further flattening to occur when heat is applied
to dry the section
 Temperature should be at the melting point of the
paraffin
 For delicate tissues the temperature is reduced
and time is prolonged

53. CUTTING HARD TISSU
ES
 Causes of cutting difficulty -
o poor-fixation
o over processing
Trobleshooting -
oProlonged soaking of the block in water
oExposing the block surface to running tap water for
30 min
oReduction in knife slant may yield result
oLastly softening agents may be used

54.  Softening agent used –
oGlycerol
oPhenol (5-10%)

55. SURFACE DECALCIFICATION
 Placing the block face down in a dish that contains
a small amount of decalcification solution
 The block is rinsed well and blotted dry
Immediate section taken since decal achieved is
limited
 In over decalcification – diagnostic material may be
compromised
 Common decalcification solutions –
o5-10% Nitric acid
o5-10% Hydrochloric acid

56. FROZEN
SECTIONS

57. FROZEN AND RELATED SECTIONS
 Used in –
1. Demonstrating soluble substances
2. For urgent diagnosis
3. In IF methods
4. In immunocytochemical methods
5. In diagnostic and research emzyme histochemistry
, where emzymes are labile

58. PRINCIPLE
 When tissue is frozen , water within the tissue
turns to ice, and in this state tissue is firm, the ice
acting as the embedding medium
THE CRYOSTAT
 Refrigerated cabinet in which a modified
microtome is housed (usually a rotary type)
 Sections are best produced from fresh unfixed
tissue

59. FREEZING OF FRESH UNFIXED
TISSUE
 Freezing should be as rapid as possible
 Slow freezing cause distortion of tissue due to ice
crystal artifact
 freezing techniques are –
1. Liquified nitrogen (-190°C )
2. Isopentane cooled by liquid nitrogen
3. Carbon dioxide ‘cardice’
4. Carbon dioxide gas
5. Aerosol sprays

60. FIXED TISSUE AND THE
CRYOSTAT
 Tissue must be absolutely fresh
 Placed in formol-calcium at 4°C for 18 hrs and
then slowly frozen
 No diagnostic importance
 To obtain accurate localisation of hydrolytic
enzymes and some antigens

61. CRYOSTAT SECTIONING
CABINET
TEMPERATURE
 Most unfixed tissue will section well between -15°
to -23°C
 Fixed tissue at -7° to -12° C
 Tissue containing large amount of water section
best at warmer temperature
 If contains large amount of fats – requires a very
cold temperature
 If chatter lines present – indication that block is
too cold

62. ANTI ROLL PLATE
 Attached to the front of microtome
 Intended to stop curling of frozen sections
 Aligned parallel to blade edge

63. ULTRAMICROTOM
Y

64. GLASS KNIVES
 Prepared from commercially available plate
glass strips measuring (40*2.5) cm
 3 diff thickness is available – 6.4, 8 and 10 mm
 2 triangular shaped knives are produced with
different cutting edge angles are produced
 Higher angle knives (upto 55°) – best for cutting
hard materials
 Shallow angles (35°) – for softer blocks

65.  When placed in ultramicrotome and viewed under
the microscope – cutting edge appears as bright line
against a dark b/g
 The left 1/3rd of cutting edge s/b smooth and used
for cutting thin sections
 The middle third – best for trimming and cutting
semi-thin sections
 A trough is attached to the knife for floating out thin
sections after they are cut

66. DIAMOND KNIVES
 Already mounted in a metal block (incorporating a
section collecting trough)
 Cutting edge must be clean – polystyrene strips
are available for this purpose

67. TROUGH FLUIDS
 Fluid used is distilled water
 10-15% solutions of ethanol or acetone may be
used
 It is important to insure the correct level of fluid is
added
 Level can be seen under the ultramicrotome
microscope as a flat, silvery-gold reflection which
should reach right to the knife edge
 Fluid can be added or removed from trough using
a fine pipette

68. PROBLEMS AND SOLUTION FOR
PARAFFIN SECTION
 Ribbon/consecutive section curved –
oBlock edge not parallel – trim the block until parallel
oDull blade edge – replace blade
oExcessive paraffin – trim away excess paraffin

69.  Thin & thick sections –
o Paraffin too soft for tissue/condition – remove
excess paraffin from blade edge
o Insufficient clearance angle – increase clearance
angle
o Faulty microtome mechanism – maintain
microtome
o Blade/block loose in holder - tighten block & blade

70.  Chatter – thick & thin zone parallel to blade
edge-oKnife/
block loose in holder – tighten blade levers
oExcessively steep clearance angle –Reduce
angle
oCalcified areas in tissue – surface decalcify
oOver hydration of tissue – Dehydrate
oDull blade – replace/use new area of blade

71. CHATTER EFFECT

72. PARAFFIN SECTIONS FORMING
RIBBON

73.  Splitting of sections at right angle to knife –
oNicks in blade – replace/use different area
oHard particle in tissue – if calcium- surface decal
oHard particle in paraffin – remove with pointed
scalpel
 Section do not form ribbon –
oParaffin too hard – re-embed in lower melting
point paraffin, warm block surface
oDebris on knife edge – clean blade & its holder
oClearance angle incorrect - adjust

74.  Section attached to block on return stroke –
oInsufficient clearance angle – increase angle
oDebris on blade edge – clean
oDebris on block edge – trim edge of block
 Incomplete section –
oIncomplete impregnation with paraffin – re-process
block
oTissue incorrectly embedded – re-embed &
correct orientation
oSection superficially cut – re-face block, cut
deeper

75.  Excessive compression –
oDull blade - Replace
oParaffin too soft – cool block face & re-cut
 Section expand or disintegrate on water bath
–
oIncomplete tissue processing – re-process
oWater temp. too high – turn down the temp.

76.  Difficulty in cutting a smooth flat section –
oWarm block face with warm water
oGentle exhale breath onto block surface during
sectioning
 Section roll into a tight coil instead of remain
flat on knife edge –
oBlade dull – replace
oRake angle too small – reduce blade tilt
oSection too thick – reduce thickness

77. CONCLUSION
 Practical experience under the guidance of a
skilled tutor is required to gain confidence & co-ordination
for microtomy
 For proper maintenance of the microtome –
oFollow manufacturers instructions
oImplement departmental policy

78. REFERENCES
1.Theory and practice of histological
techniques by John D Bancroft and Marilyn
Gamble
2. Handbook of histopathological and
histochemical techniques by C. F. A. Culling
3. Internet sources

79. T
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