impreg, embedd, section-cutting

impregnation

also known as infiltration, process where clearing agent is completely removed

25x

the impregnating medium should be atleast ___ the volume of the tissue

2-5 degree celcius above melting point of the wax

temperature required for impregnation

paraffin wax, celloidin wax, gelatin

3 types of impregnating medium

paraffin wax

simplest, most common and best embedding medium, for routine tissue processing

paraffin wax

this medium fills up the cavities and tissue spaces to give firm consistency

56 degree C

melting point of paraffin wax

54-58 C

50-54 C

MP if lab is at 20-24 C :

MP if lab is at 15-18 C :

2 C, melting point

Fresh wax should be filtered before use in a wax oven at a temperature of __ higher than its ___

excessive shrinkage and tissue hardening

what will happen if the paraffin was impregnation is prolonged

retention of clearing agent

inadequate paraffin wax impregnation promotes ___

it may be melted and filtered for future use

if the paraffin wax is trimmed away:

heating wax at 100-105°C

water present in the paraffin wax can be removed through:

twice, fresh wax

how many times paraffin was maybe used? and what will be utilized after

manual processing

automatic tissue processing

vacuum embedding

3 methods of paraffin wax impregnation

1 hour

complete method if manual processing (duration)

4 changes of paraffin at 15 mins interval

there should be atleast ____ to ensure removal of the clearing agent

automatic tissue processing

uses autotechnician

3 C above melting point of wax

wax bath temp of automatic tissue processing

constant agitation

advantage of automatic tissue processing

vacuum embedding

this method is recommended for urgent biopsies, delicate tissues

400-500 mmHg under negative atmospheric pressure

wax impregnation of vacuum embedding

vacuum impregnation

paraffin impreganation that gives the fastest result

reduces 25-75% of normal time

the time required for complete impregnation in vacuum embedding

2-4 C above melting point of wax

temperature required for vacuum embedding

paraplast

embeddol

bioloid

tissue mat

ester wax

carbowax

paraffin wax substitutes

paraplast

- highly purified paraffin and synthetic plastic polymers

- recommended for large tissue block (bones and brains)

56-57 C

melting point of paraplast

embeddol

synthetic was substitute similar paraplast but less brittle and less compressible

56-58 C

MP of embeddol

bioloid

a semisynthetic wax recommended for embedding the eyes

tissue mat a

a product of paraffin containing rubber with same property as paraplast

ester wax

can be used to the tissue even without prior clearing of the tissue specimen

46-48 C

melting point of ester wax

95% ethanol

ester wax is only soluble to ___

carbowax

the most commonly used water soluble wax

dehydration and clearing

what are skipped when carbowax is used as impregnation medium

dissolving during floatation

major disadvantage of carbowax

- pearse solution, blank & mccarthy solution

carbowax can be floated out in solutions:

diethylene glycol, distilled water, and strong formaldehyde

composition of pearse solution

gelatin and potassium dichromate

Blank and McCarthy solution composition

Celloidin Impregnation

impregnating medium for tissues with large hollow cavities that tend to collapse, for hard tissues such as TEETH and BONES for large tissue sections of the whole embryo

celloidin

infiltration agent, purified form of nitrocellulose that is soluble in alcohol and water

thin sections and sections for photomicrography

things that are hard to obtain in celloidin impregnation

wet celloidin method

dry celloidin method

nitrocellulose method

3 types of celloidin impregnation

wet celloidin method

recommended for bones, teeth, large brain sections and whole organs

70-80% alcohol

tissue block in wet celloidin method is stored at ___ to avoid dehydration to shrinkage of tissues until ready for cutting

dry celloidin method

is preferred for processing of whole eye sections

gilson's mixture

made up of chloroform and cedar wood oil to make the tissue transparent

nitrocellulose method

low viscosity nitrocellulose

nitrocellulose method

soluble in equal concentration of ether and alcohol w lower viscosity to be allowed to be used in higher concentration and can still penetrate the tissue rapidly

tissues have tendency to crack

plasticizers

problem and remedy in nitrocellulose method

gelatin impregnation

rarely used except when dehydration is avoided when tissues are subjected to histochemical and enzyme studies

gelatin impregnation

used for delicate specimens and frozen sections

25x

volume of the tissue in gelatin impregnation should be atleast ___ the volume of the tissue

2-3 mm thick

tissue in gelatin impregnation should not be more than ___

1% phenol

this prevents the growth of molds in gelatin dehydration

carbowax and gelatin

use of this will no longer require dehydration and clearing

embedding

process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify

5-10 C above its melting point

temperature of embedding required

precise position in the mold

most important step in embedding

Leuckhart's Embedding Mold

Compound Embedding Unit

Plastic Embedded Rings and Based Mold

3 molds for non disposable embedding

Leuckhart's Embedding Mold

2-L shaped strips of heavy brass arranged in a flat metal plate which can be moved to adjust the size of a mold to the size of specimen

compound embedding unir

interlocking units resting on a flat metal base

Plastic embedded rings and based mold

special stainless steel based mold fitted with plastic embedded ring

peel away

plastic ice tray

paper boats

3 disposable molds for embedding

peel away

disposable thin plastic embedding molds, available in different sizes, giving perfect even block without trimming.

plastic ice tray

recommended for busy routine laboratory, have to smear inner mold with GLYCERIN before use

paper boats

used for embedding celloidin blocks but are equally useful for paraffin blocks, glycerin will facilitate easy removal of block

double embedding method

- tissue is first infiltrated with celloidin and embedded with paraffin

- facilitate the cutting of large blocks and dense film tissues like brain

- recommended for making small sections of celloidin blocks

trimming

process of removing excess wax after embedding

2 mm of wax

in trimming, at least how much mm of wax should surround the tissue per slide?

coarse trimming

sides, tips, and bottom of the tissue are trimmed using a knife or a blade to form a truncated pyramid/4-sided prism

fine trimming

Block is placed in the MICROTOME setting the adjuster a t 15 mm or by advancing the block using the coarse feed mechanism

sectioning

also known as cutting

sectioning

process wherein a processed tissue is cut into uniformly thin slices or sections using microtome to facilitate studies under the microscope

4-6 micra

section thickness of histologic procedures

4-6 micra

thickness of paraffin section

10-15 micra

thickness of celloidin section

10 micra

thickness of frozen sections

0.5 to 1 micron

thickness of semithin section

50-120 millimicra or 500 to 1200 angstrom

ultrathin section thickness

rocking microtome (paldwell trefall)

rotary microtome (minot)

sliding microtome (adams)

ultrathin microtome

freezing microtome (queckett)

types of microtomes

rocking microtome (paldwell trefall'

cutting serial sections large block of paraffin embedded tissues, simplest among diff types

10-12 micra

rocking microtome thickness

Rotary Microtome (Minot)

cutting paraffin embedded sections, most common type used for routine and research laboratories

between 4-6 micra

rotary microtome thickness

sliding microtome (adams)

cutting celloidin embedded sections and most dangerous because of movable and exposed knife

base sledge

movable block, stationary knife

standards slicing

stationary block, movable knife

7-9 micra

sliding microtome thickness

ultrathin microtome

For cutting plastic embedded sections for electron microscopy

Freezing microtome - Queckett

for cutting unembedded frozen sections ghat releases carbon dioxide that freezes tissues

rotary microtome

Usual type of microtome inside the cryostat

-5 to -30 C near -20 C

maintaining temperature of cryostat

-18 C to -20

optimum working temperature of cryostat

cryostat

a freezing chamber for tissue samples

fluorescent antibody staining and histochemical enzyme studies

cryostat prepares thin sections of fresh frozen tissues for:

-leave the slide inside the incubator for 37 C overnight

-place in the oven at 50-60 C for 2hrs

- place slide on top of the hot plate at 45-50 C for 30-45 mins

ways to dry ribbon

25mm

length of plane concave