CUTTING OF PARAFFING WAX EMBEDDED SECTIONS
TRIMMING THE BLOCK
It is the process of cutting or removing excess wax from the block to expose tissue
for sectioning.
While trimming at least 2-2.5 mm wax is left on all four sides of the tissue.
The tissue becomes exposed
FIXING THE BLOCK ON THE MICROTOME
The rear end of the tissue block is just touched on the surface of hot plate or Bunsen
burner and quickly attached to a wooden or metal block holder.
CUTTING/SECTIONING TECHNIQUE
Successful sectioning requires properly prepared tissue block, sharp knife, proper
microtome, a skilled technician
Place the microtome on a steady table and the operator should be seated at a
convenient height in front of it.
Insert a microtome knife/blade in the knife holder and screw it tightly in position.
The block is fixed on to the clamp type holder on microtome.
Adjust the clearance angle between 2-6 degree.
Move the block holder forward using the handwheel so that the block is almost
touching the edge of the knife
Tighten all the adjusting screws
Set the section thickness gauge at 4-5 micrometre thickness.
Start cutting sections with the right hand operating the microtome(handwheel) and
the left hand holding sections with forceps or camel hairbrush.
The selected sections are floated on a warm water bath and on to a slide.
MICRO GLASS SLIDES
They maybe plain or frosted
Size-75 mm long,25 mm wide
Frosted glass have an advantage over plain slide because the identification number
can be written on the frosted area by lead pencil.Plain slide needs diamind pencil to
etch the identification number .
FIXING THE SECTIONS TO SLIDES(floatation)
This is the process of removing creases from the tissue section before attaching theme to
slide.
Methods:
Water bath method
, Hot stage method
Warmed slide method
WATER BATH METHOD
Thermostatically controlled water baths are used.
The inside of the water bath is black coloured and temperature is controlled 5-10
degree below the melting point of wax
The water bath should be 25-30 cm in diameter and 8-10 cm depth.
The tissue sections are divded with a scalpel into lengths that go on a slide(usually
single section or 3-4 small sections)
The tissue sections are lifted off the microtome and transferred on to the surface of
water.
Using camel hair brush one end of the tissue section is gently lowered into the water
bath and gradually the other end is lowered.
Mild stretching force is applied to remove the creases using forceps
Once the folds have disappeared ,a clean slide is dipped obliquely into water bath
close to the section and then gradually withdraw the slide so that the whole section
is transferred to the slide.
Place the slide in upright position to drain excess water
It is then kept in incubator at 37 degree
Causes of persistence of creases
Block was not sufficiently cold or there is defect in knife
Disadvantages of water bath method
In disease condition such as leprosy ,the bacilli from one section is transferred to te
subsequent sections as floaters and may lead to erraneous results.
HOT STAGE /PLATE METHOD
Hot stage consist of a specially shaped metal top heated by electricity to maintain
the temperature of the stage at 45-50 degree
A clean slide is placed on to the hot stage,flooded with distilled water.
The sections are floated on the surface of water.
Under the influence of heat small creases will disappear and the section will flatten
out.When the section is completely flat,remove the slide from the hot plate and
excess water is drained off
WARM SLIDE METHOD
A clean slide is flooded with distilled water and sections are placed on the slide
After major creases removed,the slide is held over Bunsen burner for 1-2 seconds ,if
needed a second heating may be given.
Remove the excess water and keep the slide into oven at a temperature of 56 degree
for drying.
TRIMMING THE BLOCK
It is the process of cutting or removing excess wax from the block to expose tissue
for sectioning.
While trimming at least 2-2.5 mm wax is left on all four sides of the tissue.
The tissue becomes exposed
FIXING THE BLOCK ON THE MICROTOME
The rear end of the tissue block is just touched on the surface of hot plate or Bunsen
burner and quickly attached to a wooden or metal block holder.
CUTTING/SECTIONING TECHNIQUE
Successful sectioning requires properly prepared tissue block, sharp knife, proper
microtome, a skilled technician
Place the microtome on a steady table and the operator should be seated at a
convenient height in front of it.
Insert a microtome knife/blade in the knife holder and screw it tightly in position.
The block is fixed on to the clamp type holder on microtome.
Adjust the clearance angle between 2-6 degree.
Move the block holder forward using the handwheel so that the block is almost
touching the edge of the knife
Tighten all the adjusting screws
Set the section thickness gauge at 4-5 micrometre thickness.
Start cutting sections with the right hand operating the microtome(handwheel) and
the left hand holding sections with forceps or camel hairbrush.
The selected sections are floated on a warm water bath and on to a slide.
MICRO GLASS SLIDES
They maybe plain or frosted
Size-75 mm long,25 mm wide
Frosted glass have an advantage over plain slide because the identification number
can be written on the frosted area by lead pencil.Plain slide needs diamind pencil to
etch the identification number .
FIXING THE SECTIONS TO SLIDES(floatation)
This is the process of removing creases from the tissue section before attaching theme to
slide.
Methods:
Water bath method
, Hot stage method
Warmed slide method
WATER BATH METHOD
Thermostatically controlled water baths are used.
The inside of the water bath is black coloured and temperature is controlled 5-10
degree below the melting point of wax
The water bath should be 25-30 cm in diameter and 8-10 cm depth.
The tissue sections are divded with a scalpel into lengths that go on a slide(usually
single section or 3-4 small sections)
The tissue sections are lifted off the microtome and transferred on to the surface of
water.
Using camel hair brush one end of the tissue section is gently lowered into the water
bath and gradually the other end is lowered.
Mild stretching force is applied to remove the creases using forceps
Once the folds have disappeared ,a clean slide is dipped obliquely into water bath
close to the section and then gradually withdraw the slide so that the whole section
is transferred to the slide.
Place the slide in upright position to drain excess water
It is then kept in incubator at 37 degree
Causes of persistence of creases
Block was not sufficiently cold or there is defect in knife
Disadvantages of water bath method
In disease condition such as leprosy ,the bacilli from one section is transferred to te
subsequent sections as floaters and may lead to erraneous results.
HOT STAGE /PLATE METHOD
Hot stage consist of a specially shaped metal top heated by electricity to maintain
the temperature of the stage at 45-50 degree
A clean slide is placed on to the hot stage,flooded with distilled water.
The sections are floated on the surface of water.
Under the influence of heat small creases will disappear and the section will flatten
out.When the section is completely flat,remove the slide from the hot plate and
excess water is drained off
WARM SLIDE METHOD
A clean slide is flooded with distilled water and sections are placed on the slide
After major creases removed,the slide is held over Bunsen burner for 1-2 seconds ,if
needed a second heating may be given.
Remove the excess water and keep the slide into oven at a temperature of 56 degree
for drying.
