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The following points highlight the four methods of embedding plant tissues in paraffin blocks. The methods are: 1. Fixation 2. Washing 3. Dehydration 4. Infiltration.
Method # 1. Fixation:
(a) Flower buds:
Flower buds of suitable size are rinsed in Carnoy’s fluid for 5 sec. and then fixed in Nawaschin’s fixative (Belling’s modification) for 24 hours. Nawaschin A and B are mixed in equal proportions just before fixing. Flower buds of suitable size means buds which are likely to show divisional stages.
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To be sure of having divisions an anther may be taken out carefully from a bud and tested by the carmine smear technique to see whether it is in the dividing stage. The bud containing the remaining anthers may then be fixed. Carnoy’s fluid is used to remove the siliceous particles sometimes present on the outer surface of the sepals.
This ensures easy penetration of the fixative into the bud. Treatment in Carnoy’s fixative is restricted to a few seconds because Carnoy’s fixative has a rapid penetrating capacity. So prolonged treatment in Carnoy’s fluid implies that the tissue will become fixed in Carnoy’s rather than Nawaschin’s fixative.
(b) Root-tips:
Take actively growing fresh root-tips about 1 cm long after washing them thoroughly in running water to remove all soil particles, and fix them in Liwitsky’s fixative (1% chromic acid and 10% formalin mixed in 1 : 1 ratio just before use) for 24 hours. Both Nawaschin’s and Liwitsky’s fixatives are brownish in colour. But after mixing the colour gradually changes to greenish indicating the reduction of chromic acid.
Method # 2. Washing:
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After fixation the material is washed in running water for 24 hours. It can also be washed for 3 hours in tepid warm water changing the water every half-hour. Washing is done to remove the excess fixative from the material which may interfere with staining later.
Method # 3. Dehydration:
Dehydration is done by ethyl alcohol. Pass through 30% and 50% alcohol for 1 hour each; 70% alcohol for overnight (if required the material can be preserved in 70% alcohol for an indefinite period); 80%, 90% and 95% alcohol for 1 hour each; absolute ethyl alcohol for overnight and a second change for 10 minutes.
Method # 4. Infiltration:
Pass through the following grades: absolute ethyl alcohol — chloroform (3: 1), (1: 1) and (1: 3) for 1 hour each and then pure chloroform — giving two changes of 10 minutes each.
Keep a little chloroform in the tube, add paraffin chips of low melting point and keep the tube on a hot plate at 37°C for 48 hours, removing the cork after 24 hours. Transfer the tube to a 45°C paraffin bath and keep overnight. The tube can now be stored at room temperature.
For microtome sectioning the tissue is embedded in paraffin while it is fixed in aqueous fixative. Therefore, after fixation, the water is to be replaced by a medium in which paraffin is soluble, such
as chloroform or xylol. While cytologists prefer chloroform, anatomists use xylol. But these are not miscible with water. So water is first replaced by alcohol.
Alcohol is miscible with both water and chloroform or xylol. Finally, alcohol is replaced by chloroform or xylol and in this medium paraffin is added. This process, called Cleaning, is always done gradually by using a graded series so that plasmolysis or mechanical distortion does not occur.
