Impregnation and Embedding

Question 1

Process whereby the clearing agent is completely removed from the tissue & replaced by a medium that will completely fill all the tissue cavities, thereby giving a firm consistency to the specimen

Answer 1

Impregnation

Question 2

_________ (Casting or Blocking) is the 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

Answer 2

Embedding

Question 3

Characteristics of an Ideal infiltrating and embedding medium

Answer 3

• soluble in processing fluids
• suitable for sectioning and ribboning
• molten between 30°C and 60°C
• translucent or transparent; colorless
• stable
• homogeneous
• capable of flattening after ribboning
• non-toxic
• odorless
• easy to handle
• inexpensive

Question 4

Tissue Impregnation

Answer 4

• Paraffin wax Impregnation
• Celloidin Impregnation
• Gelatin Impregnation
• Plastic

Question 5

• _________ is the simplest, most common and best embedding medium used for routine tissue processing.
• __________ is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils.
• It is solid at room temperature but melts at temperatures up to about 65°C or 70°C.

Answer 5

Paraffin Wax Impregnation

Question 6

Paraffin wax advantages:

Answer 6

1. Thin individual serial sections may be cut
   with ease
2. The process is very rapid
3. Tissue blocks and unstained mounted
   sections may be stored in an indefinite
   period of time
4. Formalin-fixed, paraffin-embedded
   tissues may be stored indefinitely at room
   temperature,
5. Many staining procedures are permitted
   with good results.

Question 7

Paraffin wax disadvantages:

Answer 7

1. Overheated - brittle.
2. Prolonged impregnation - tissue shrinkage and hardening
3. Inadequate impregnation will promote retention of the clearing agent. - Tissues become soft and shrunken,
4. Tissues that are difficult to infiltrate, need long immersion for proper support
5. Paraffin processing is not recommended for fatty tissues.

Question 8

• Common waxes have melting points of:
• _____________________________.
• ______ wax is normally used for routine work.
• melting point of ________ - In a laboratory with temperature ranging from _______.
• Between ___________. If the laboratory temperature is between _________.
• Hard tissues require wax with a higher melting point than soft tissues.

Answer 8

• Temperature
• 45°C, 52°C, 56°C and 58°C
• 56°C
• 54-58°C
• 20-24°C
• 50 and 54°C
• 15-18°C

Question 9

Three ways by which Paraffin wax impregnation and embedding may be performed

Answer 9

• Manual Processing
• Automatic Processing
• Vacuum Embedding

Question 10

Manual Processing

Answer 10

• At least four changes of wax are required at 15 minutes intervals. in order to insure complete removal of the clearing agent from the tissue.
• The specimen is then immersed in another fresh solution of melted paraffin for approximately 3 hours to insure complete embedding or casting of tissue.

Question 11

• Fixation : 10% Buffered Formalin
• Dehydration : 70% Alcohol
• 95% Alcohol
• 100% Alcohol
• 100% Alcohol
• 100% Alcohol
• Clearing : Xylene or Toluene
• Impregnation : Paraffin wax
• Embedding : Paraffin wax

Answer 11

• 24 hours
• 6 hours
• 12 hours
• 2 hours
• 1 hour
• 1 hour
• 1 hour (2x)
• 15 minutes (4x)
• 3 hours

Question 12

Automatic Processing

Answer 12

• This method makes use of an automatic tissue
processing machine (i.e., Autotechnicon) which fixes, dehydrates, clears and infiltrates tissues.
• This results in a more rapid diagnosis with less
technicality.
• Usually, only 2- 3 changes of wax are required to remove the clearing agent

Question 13

• 10% Buffered Formalin
• 10% Buffered Formalin
• 70% Alcohol
• Absolute Alcohol (1)
• Absolute Alcohol (2)
• Absolute Alcohol (3)
• Xylene or Toluene (1)
• Xylene or Toluene (2)
• Benzene (1)
• Benzene (2)
• Chloroform (1)
• Chloroform (2)
• Chloroform (3)
• Wax (1)
• Wax (2)

Answer 13

• 2 hrs.
• 2 hrs.
• 2 hrs.
• 1 hr.
• 1 hr.
• 1 hr.
• 1 hr.
• ## 1 hr.-
  -
  -
  -
• 2 hrs.
• 3 hrs.

Question 14

• 10% Buffered Formalin
• 10% Buffered Formalin
• 70% Alcohol
• Absolute Alcohol (1)
• Absolute Alcohol (2)
• Absolute Alcohol (3)
• Xylene or Toluene (1)
• Xylene or Toluene (2)
• Benzene (1)
• Benzene (2)
• Chloroform (1)
• Chloroform (2)
• Chloroform (3)
• Wax (1)
• Wax (2)

Answer 14

• 2 hrs.
• 2 hrs.
• 2 hrs.
• 3 hrs.
• 3 hrs.
• ## 3 hrs.-
• 30 mins.
• ## 1 hr.-
  -
• 3 hrs.
• 3 hrs.

Question 15

• 10% Buffered Formalin
• 10% Buffered Formalin
• 70% Alcohol
• Absolute Alcohol (1)
• Absolute Alcohol (2)
• Absolute Alcohol (3)
• Xylene or Toluene (1)
• Xylene or Toluene (2)
• Benzene (1)
• Benzene (2)
• Chloroform (1)
• Chloroform (2)
• Chloroform (3)
• Wax (1)
• Wax (2)

Answer 15

• 2 hrs.
• 2 hrs.
• 2 hrs.
• 2 hrs.
• 2 hrs.
• 2 hrs.
• ## --
• 2 hrs.
• 2 hrs.
• 2 hrs.
• 2 hrs.
• 2 hrs.

Question 16

• Changed of solutions will depends on the number and sizes of the tissues processed.
• Dehydrating fluids should be changed frequently
• The clearing agent and the dilute ethanols should be changed at least once a week.
• To avoid spillage, fluid and wax containers must be filled to the appropriate level and correctly located in the machine.
• Wax accumulating on any surface or beaker leads must be remove
• Wax bath thermostats should be set at least 3 degrees above the melting point of the wax, and timing should be checked

Answer 16

Precautions with Automatic Processing

Question 17

• ____________ involves wax impregnation under negative atmospheric pressure inside an embedding oven.
• It reduces the time when tissues are subjected to high temperatures
• It facilitates complete removal of transition solvents, and prolongs the
life of wax by reducing solvent contamination.
• ________ hastens the removal of air bubbles and clearing agent from the tissue block, thereby promoting a more rapid wax penetration of the tissue.
• This technique is particularly recommended for urgent biopsies
• With ___________, the time required for complete impregnation is reduced by 25% -75% of the normal time required for tissue processing

Answer 17

Vacuum Embedding

Question 18

• The tissue should not be left in the paraffin oven for more than 4 hours.
• The paraffin oven must be maintained at a temperature 2 to 5°C above the melting point of paraffin to be used for impregnation.
• Paraffin wax must be pure
• Fresh wax should be filtered before use
• Paraffin wax may be used only twice
• When using an automatic tissue processing machine, wax usually becomes admixed with the clearing agent
• For fixed knife microtomes, a relatively hard wax with a higher melting point is recommended.

Answer 18

Practical Considerations

Question 19

Factors Affecting Paraffin Wax Impregnation

Answer 19

• Nature and size of the tissues to be processed
• Type of clearing agents to be used
• Benzene and Xylene - easily removed
• Chloroform and cedarwood oil - difficult to remove

Question 20

Substitutes for Paraffin Wax

Answer 20

• Paraplast
• Ester wax

Question 21

• Mixture of highly purified paraffin and synthetic plastic polymers
• Melting point of 56-57°C

Answer 21

Paraplast

Question 22

Paraplast

Answer 22

• Embeddol
• Bioloid
• Tissue mat

Question 23

semisynthetic recommended for embedding eye

Answer 23

Bioloid

Question 24

melting point 56-58°C

Answer 24

Embeddol

Question 25

product of paraffin, containing rubber, same property as paraplast

Answer 25

Tissue mat

Question 26

- Has lower melting point 46-48°C - Harder than paraffin - Not soluble in water but soluble in 95%EA, can be used for impregnation without prior clearing of tissue - ( Cellosolve or xylene)

Answer 26

Ester wax

Question 27

Mostly polyethylene glycols Melting point 38-42°C or 45-56°C

Answer 27

Water Soluble waxes

Question 28

– most common (hydroscopic) it appears solid at RT. - does not require dehydration and clearing - The tissues are fixed, washed out - transferred directly to the melted carbowax 4 changes of carbowax for routine processing, one each in 70%, 90%, and 2x in 100% , at a Temp of 56°C at 30 minutes 45 minutes and 1 hour (with agitation) - suitable for many enzyme histochemical studies *** tissue sections are difficult to float and mount

Answer 28

Carbowax

Question 29

added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.

Answer 29

Dimethyl sulphoxide (DMSO)

Question 30

is a purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which tend to collapse

Answer 30

Celloidin (Collodion)

Question 31

Celloidin Impregnation Advantages:

Answer 31

1. It permits cutting of tissue sections which are thicker than in paraffin wax, and is recommended for processing of neurological tissues. 2. Its rubbery consistency allows tissue blocks that are either very hard 3. Dense tissues which are hard to infiltrate (e.g. bones and brain) and specimens which tend to collapse easily due to air spaces (e.g. eyes) are supported better. 4. It does not require heat during processing.

Question 32

Celloidin Impregnation Disadvantages:

Answer 32

1. Celloidin impregnation is very slow (lasting for several days or weeks). 2. Very thin sections (less than I 0 μ) are difficult to cut. 3. Serial sections are difficult to prepare. 4. Vapor of the ether solvent is very flammable 5. Photomicrographs are difficult to obtain. 6. It is very volatile

Question 33

2 Methods for Celloidin Impregnation

Answer 33

- Wet Celloidin Method - Dry Celloidin Method

Question 34

is recommended for bones, teeth, large brain sections and whole organs. The tissue is then placed in thin celloidin (2-4%) for 5-7 days, transferred to medium celloidin (4-6%) for another 5-7 days, drained off and poured with thick celloidin (8-12%) until the specimen has become impregnated, usually between 3-5 days. The tissue block is then stored in 70-80% alcohol until ready for cutting.

Answer 34

Wet Celloidin Method

Question 35

is preferred for processing of whole eye sections. The principle and procedure of this method is similar to wet celloidin method, except that 70% alcohol is not used for storage before cutting. Instead, Gilson's mixture, made up of equal parts of chloroform and cedarwood oil, is added to the celloidin block before hardening, to make the tissue transparent.

Answer 35

Dry Celloidin Method

Question 36

• ________________ is another form of celloidin soluble in equal concentration of ether and alcohol, with a lower viscosity. • It forms a harder tissue block and makes cutting of thinner sections • possible. • The tendency of tissues to crack may be prevented by adding plasticizers (e.g.oleum ricini or castor oil) when embedding chrome-mordanted tissues. • is more explosive than celloidin

Answer 36

- Nitrocellulose Method - Low Viscosity Nitrocellulose (L.V.N.)

Question 37

is rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies. It is used as an embedding medium for delicate specimens and frozen tissue sections

Answer 37

Gelatin impregnation

Question 38

After impregnation, the tissue is placed into a mold containing the embedding medium and this medium is allowed to solidify.

Answer 38

Embedding

Question 39

the process by which a tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining.

Answer 39

Orientation

Question 40

Embedding Molds

Answer 40

1. Leuckhart’s embedding mold 2. Compound embedding Unit 3. Plastic embedding rings & base molds - Tissue tek 4. Disposable embedding molds - Peel-away - Plastic Ice trays - Paper Boats

Question 41

OTHER EMBEDDING METHODS

Answer 41

- Celloidin or Nitrocellulose Embedding Method - Double-Embedding

Question 42

used to be recommended for embedding hard tissues such as bones and teeth, and for large sections of whole organs. - Tissues were embedded in shallow tins of enamel pans which covered by sheetts of weighted glass - Bell jars were used to control the rate of the evaporation of the solvent

Answer 42

Celloidin or Nitrocellulose Embedding Method

Question 43

is the process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with paraffin wax in which they are subsequently embedded. - This is used to facilitate cutting of large blocks of dense firm tissues like the brain. - They are also recommended for making small sections of celloidin blocks.

Answer 43

Double-Embedding

Question 44

The introduction of ____________ media has provided superior results for light microscopic studies, particularly in hard tissues such as undecalcified bone and for high resolution light microscopy of tissue sections thinner than the usual 4-6 μm, such as renal biopsies a n d bone marrow biopsies. Plastics are classified into epoxy, polyester, or acrylic, based on their chemical composition.

Answer 44

PLASTIC (RESIN) EMBEDDING

Question 45

• made up of balanced mixture of epoxy plastic, catalysts and accelerat

Answer 45

Epoxy embedding plastic

Question 46

Three types are used in microscopy

Answer 46

- bisphenol A (Araldite) - glycerol (Epon) - cyclohexene dioxide (Spurr)

Question 47

slow infiltration

Answer 47

bisphenol A (Araldite)

Question 48

lower viscosity

Answer 48

glycerol (Epon)

Question 49

have very low viscosity, and infiltrates faster

Answer 49

cyclohexene dioxide (Spurr)

Question 50

were originally introduced for EM in the mid-1950s, but have been superseded by more superior epoxides, and are now seldom used.

Answer 50

Polyester plastic

Question 51

made up of esters of acrylic or merthacrylic acid, and are used extensively for light microscopy

Answer 51

Acrylic plastics