Technovit® 9100 Methyl Methacrylate Application Information from Heraeus Kulzer

EMS Catalog #14655

Areas of Application:

Technovit®9100 New is a polymerization system based on methyl methacrylate and has been specially developed for embedding mineralized tissues for light microscopy.

1. Heavy duty microtome technique for preparing thin sections: for example, pelvic biopsies, small dense bone samples.
2. Cutting-grinding and micro-grinding system (contact point technique): for example, jaw and teeth segments with and without implant.
3. Thin and semi-thin section technique for soft tissues: for example liver, kidney, testicular biopsies and lymph nodes.
4. Combined contact point and heavy duty microtome techniques: for example, interface regions and examination of tissues surrounding metal implants and cement-free endoprostheses (target tissue preparation).

Properties:

The chemical polymerization of Technovit® 9100 New takes place under the exclusion of oxygen with the aid of a catalyst composed of a peroxide and an amine component.  Additional components such as PMMA-powder and a regulator allow for a controlled polymerization by temperatures between –8 and –20°C, which guarantee a dispersion of the heat generated during polymerization.  The polymerization time is between 15 and 24 hours using a volume between 3 and 15ml for the above temperature range.  After necessary pretreatment of the tissue – fixing, dehydrating and defatting as well as pre-infiltration, infiltration and polymerization-all current staining methods can be applied to the MMA-sections.  These include classical staining as well as immuno- and enzyme-Histochemistry.  In-situ hybridization (ISH) techniques can also be performed.

Table 1: Components of the Technovit®9100 New polymerization system:

Component	Amount	Component No.
Basis Solution, (stabilized)	1 litre	1
PMMA-powder	120g	2
Hardener 1	8 sachets á 1 g	3
Hardener 2	10ml	4
Polymerization regulator	5ml	5

The basis solution Technovit®9100 New can be used in stabilized or destabilized form.  The use of destabilized Technovit®9100 New basis solution guarantees results for all immunohistochemical methods analogous to those using paraffin sections.

Destabilizing the Basis Solution:

Fill chromatography column ¾ with Aluminum Oxide 90 Basic (Al₂O₃) EMS# 10400 and allow the Technvit®9100 New basis solution (component no.1) to flow slowly through it.  A column prepared as above is sufficient to destabilize 3-4 litres of basis solution.  Store the destabilized basis solution in portions in stoppered brown glass bottles and store either at 4°C for up to one week or at –15°C to –20°C for up to two weeks

Table 2: Processing Procedures

Component Number	1	2	3	4	5
Component	Basic Solution	PMMA Powder	Hardener 1	Hardener 2	Regulator	Storage
Pre infiltration	200ml 190g		1 g			7 days at RT
Infiltration	ad 250ml 237.5g	20 g	1 g / 2 g*			7 days at 4°C
Stock solution A	ad 500ml 475g	80 g	3 g / 4 g*			7 days at 4°C 14 days at -20°C
Stock solution B	ad 50ml 47.5g			4 ml	2 ml	7 days at 4°C 14 days at -20°C
Polymerization mixture	9 parts (v/v) Stock Solution A Plus 1 part (v/v) Stock solution B					Use immediately

Important: **When using stabilized Technovit® 9100New the increased amounts (2g ad 250ml and 4g ad 500ml basis solution) of Hardener 1 must be used.

The time needed for pre-infiltration is at least 1 h at room temperature and up to 24h depending on the size of the specimen. For infiltration at 4°C the time taken is at least 1 h up until 24 h or longer.  In the case of large specimens such shared bones of endoprosthetics 5 up to 7 days at 4°C is needed for complete infiltration.

Preparation of stock Solution A:

80g powder (component number 2) is added stepwise to a 500ml graduated measuring cylinder containing ca. 400ml stabilized or destabilized basis solution (component number 1) using a magnetic stirrer.  When solution has been achieved, 3g (or 4g – see above) hardener 1 (component number 3) is added under further stirring. Finally, basis solution is added until the 500ml graduation mark has been reached. The resulting stock solution A, which is characterized through its viscosity should be transferred to a screw-top brown glass bottle, labeled as Stock Solution A and stored at either 4°C or –20°C (see above) until use.

Preparation of stock Solution B:

4ml hardener 2 (component number 4) is added by pipette to a 50ml graduated measuring cylinder containing ca. 30ml stabilized or destabilized basis solution (component number 1) on a magnetic stirrer. 2ml regulator (component number 5) are then pipetted and the solution made up to 50ml (calibration mark) using basis solution and the resulting mixture stirred thoroughly.  The mixture, which is not viscous, should then be transferred to a screw-top brown glass bottle, labeled as Stock Solution B and stored at either 4°C or –20°C until use.

NB: the regulator solution must be added from a pipette!

Preparation of the polymerization mixture:

Cooled stock solutions A & B should be mixed in the proportion 9 parts A (measuring cylinder) plus 1 part B (pipette) directly before use.  This should be performed in a beaker using a glass rod to stir the mixture.  The samples are then positioned in containers and completely covered with polymerization mixture, placed in a cooled dessicator and a partial vacuum produced – for example using a water pump – and left to stand at 4°C for ca. 10 minutes.  The resulting blocks are then placed in a sealed container and left to polymerize at between –8°C and –20°C.

Embedding and Polymerization:

• Embedding form must be filled up ENTIRELY with mixture of storage solutions A & B
• Generate a vacuum around the mold (max. 200mbar – low vacuum), +4°C approx. 10 minutes.
• Close embedding mold ABSOLUTELY TIGHT; make sure that no air is inside the embedding mold
• Put the mold into the refrigerator

Polymerization:

The polymerization process takes place at –8°C to –20°C.  The samples are then allowed to stand at 4-8°C (refrigerator) for at least one hour before being allowed slowly to come to room temperature.

The polymerization times are dependent on the volumes of polymerization mixture used and of the constancy of the temperature at which polymerization is carried out.  Larger samples should be polymerized at lower temperatures.  The cooling capacity of the refrigerator (ice box, deep freezer, freezer-unit with lid) and the volume of polymerization solution should be taken into account when preparing for polymerization.  Here are some examples:

	Gelatin capsules, approx. 0.2ml	-2°C to –4°, 24 h
	PTFE Molds, approx. 3ml	-2°C to –4°, 24 h
	PVC Capsules, approx. 15ml	-3°C to –4°C, 24 h
	PVC molds 200-300ml	-10°C to – 15°C, 24 h

 

When the prepared samples have been brought to ambient temperature, they can be mounted on blocks using Technovit®3040 in order to remove the samples from the PTFE mold.

Further preparation:

When the blocks have been mounted they can be cut.  Before cutting, the block must be wetted with 30% ethanol.  The sections are removed and the sections are removed and placed on precoated slides using 50% age ethanol. Finally the sections are covered with polyethylene foil, pressed at 37°C – 60°C overnight and then deplasticized before being stained.

Necessary information about fixing, dehydrating, deplasticizing, routine staining and immunohistochemistry, as well as procedures for different tissues is available by clicking here.

Danger Advice:

Basis Solution, Polymerization regulator:	Irritating to eyes, respiratory system and skin. May cause sensitization by skin contact.
Hardener 1:	May cause fire.  Irritating to eyes. May cause sensitization by skin contact.
Hardener 2:	Harmful by inhalation, in contact with skin and if swallowed.  Danger of cumulative effects.  Irritating to eyes, respiratory system and skin.

Safety Advice:

Basis Solution:	Keep container in a well-ventilated place.  Keep away from sources of ignition – No smoking.  Avoid contact with skin. In case of contact with eyes rinse immediately with plenty of water and seek medical advice.  Wear suitable gloves.
Hardener 1:	Keep container tightly closed in a cool place.  Keep away from reducing agents, heavy metal compounds, acids and alkalis.  In case of contact with eyes rinse immediately with plenty of water and seek medical advice.  Wear suitable protective clothing and gloves.
Hardener 2:	Keep container in a well-ventilated place.  Keep away from acidic substances and heavy metal compounds.  In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.  Wear suitable protective clothing and gloves.

Source of Information

Heraeus Kulzer, 2010

Online Ordering

Technovit® 9100 Methyl Methacrylate is available online from the EMS Catalog. For ordering or product information, click here.