Embedding

Microscopy Methods in Microbiology
Microroscopy Methods in Microbiology Part 2
Microroscopy Methods in Microbiology - Introduction
Observation of Microbes in the Living State
Observation of Microbes in Smears
Dyes and Staining
Useful pH Range and Colour Change of Some Indicator Dyes
Factors Affecting Staining
Microscopy and Micrometry
Microscopy and Micrometry - Introduction
Behaviour of Light
Light Theories
Resolving Power
Refraction and Reflection
Bright Field Microscopy
Use of Bright Field Microscope

Embedding

Embedding:
The embedding process for electron microscopy is quite different than for light microscopy. Paraffin wax does not impart sufficient strength, to the material to maintain in structure, during the section cutting with the help of ultramicrotome. Less than 0.1 μm thick sections have been obtained when high polymer plastics and resins are used as embedding medium.

The presence of plastic embedding material prevents the collapse of the specimen. The most frequently used materials are epoxide resins like; Epon, Araldite and Maraglas. Extreme care is necessary in handling these materials because they may have carcino genic effect and are known to cause skin reactions.

Fixed bacterial suspension is processed in the following way before embedding into the plastic material. Mix 0.03 ml of 2% molten agar kept at 450C to the bacterial suspension. Pour a drop of molten agar suspension on a clean microscope, slide and allow it to set. Cut the agar block of 1 mm size with the help of razor blade. Treat the block with 0.5% uranyl acetate solution for two hours at room temperature. Dehydrate the block and use for embedding.

Embedding is generally carried out in gelatin capsules of various sizes. According to Luft (15) Epon mixture can be prepared by ,mixing solution-A (S4) and solution-B (S5) in varying proportion according to the degree of hardness required as shown in Table 2-II.

Preparation of Epon embedding mixture:

No.	Hardness of final block	Solution-A (ml)	Solution - B (ml)
1.	Soft	80	20
2.	Standard	60	40
3.	Medium	50	50
4.	Hard	30	70

During mixing of A and B solutions, 2% of benzyl-dimethyl amine is added to speed up the process of polymerisation. Gelatin capsuIe is filled almost to the top with embedding material and the material to be embedded is kept into the capsule and allow it to sink slowly through the viscous fluid. The polymerisation of material can be accomplished by keeping the reparation in an oven adjusted at 60°C for 24 to 48 hours.

Araldite mixture has also similar properties. Mercer and Birbeck (16) recommended following composition of Araldite mixture. Dodecelyl succinic anhydride, 20 ml; Araldite M, 20 ml; 2,4,6 -trimethylaminomethyl phenol (DMP-30), 0.6 ml and dibutylphthalate, 2.0 ml. The mixture is heated to 50°C and after thorough mixing can be used as described for the Epon.

Vestopal introduced by Ryter and Kellenberger (17), is a polyester resin and has similar characteristic to Araldite. Maraglas epoxy resin introduced by Freeman and Spurlock (18) is also employed in biology.

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