FORM 2
THE PATENT ACT 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION- PARAFFINATION TECHNIQUE FOR PREPARATION OF
MUSEUM SPECIMEN BY IMPREGNATION OF PARAFFIN WAX IN TISSUE.
2. APPLICANT(S)
(a) NAME: PATH SUMITTULASHIDAS
(b) NATIONALITY:- INDIAN
(c) ADDRESS:- c/o T.S. Patil, "Advaif Ram Nagar, Teacher Colony, Jafrabad Road,
At/Po CHIKHLI, Dist- Buldana Maharashtra Pin- 443201
3. PREAMBLE TO DESCRIPTION
COMPLETE
The following specification describes The invention and the manner in which it is to be performed
4. DESCRIPTION (Description shall start from next page)

4. DESCRIPTION
PARAFFI NATION TECHNIQUE FOR PREPARATION OF MUSEUM SPECIMEN BY IMPREGNATION OF PARAFFIN WAX IN TISSUE.
By the paraffination technique, water from the biological specimen (tissue) is replaced by paraffin wax so that autolysis and bacterial decomposition stops. Specimen so prepared can be used for educational purpose in medical, veterinary and science fields.
Plastination is a technique of body tissue preservation with a great variety in its processes and development which is originally introduced to the medical world by Dr. Gunther von Hagens in 1977 at New York, USA. In these processes, water and lipids in biological tissues are replaced by curable polymers mostly silicone, epoxy and polyester which then will harden and finally result in natural looking, dry, odourless and durable specimens. In the process of plastination specimens were flushed with tap water, prepared and fixed with formalin. After fixation they were dehydrated in cold acetone (-15 to -25°C) with three weekly changes. Acetone saved stabilisation of shape and caused only minimal shrinkage of tissue. The dehydrated specimens were impregnated at room temperature with a polymer reaction-mixture of silicon S 10 and S 3 (100: 1). The final vacuum ranged between 2 and 15 mm Hg. The process was completed after eight weeks impregnation. The specimens were drained of ex- cess of polymer and hardened by exposure to UV light for two days. After hardening, the specimens were applicable in teaching process.
India is a developing country and to date the preservation of biological tissues is done by conventional methods such as perfusion and embalming. The chemical commonly used is formaldehyde which is known to be toxic. In these methods the penetration of chemicals into deeper parts of the organ or body depends on the flow of fixatives by perfusion pressure through blood

vessels. If tissues are well perfused the cells are fixed in situ. Tissues are also preserved by immersion whereby they are kept in the fixatives for long preservation. Organs used for study are always kept moist by fixative solution and user has to wear gloves for protection of hand. Plastinated specimens are not used by all in India due to costly technique and specimens. So paraffination method is developed as an alternative method of plastination to prepare handy, cost-effective, odourless specimens of tissue/organs.
EXPERIMENTAL PROCEDURE - Paraffination technique includes tissue or specimen collection, tissue fixation, dehydration, clearing, paraffin impregnation and finishing.
Tissue/specimen collection- Tissue or organ required for the paraffination technique was collected from the bodies donated to the department of Anatomy of Government medical college, Nagpur. The tissue/organ collected was dissected properly for visualisation of required structure. Tissue can also be obtained from operation theatre, mortuary or animal tissue.
Fixation - In the process of fixation, the fixative causes coagulation of tissue proteins and constituents thus minimise their loss or diffusion during processing. 10 % formalin solution is most commonly used for fixation. The specimen is kept in 10 % formalin for 10 - 15 days for fixation as per size. Mercuric chloride- formalin, Susa fixative, Zenker's fluid, Bouins's fluid, Kaiserling solution can be used for fixation. Fixative solutions are prepared by using formaldehyde, glutaraldehyde, mercuric chloride, potassium dichromate, chromic acid, picric acid, osmic acid or acetic acid.
Dehydration- Paraffin wax will not penetrate tissue in the presence of water, so dehydration is essential. This is effected by immersion of tissue in ethyl alcohol of ascending grade from 70%, 90% and then absolute alcohol. For

200 gram of specimen minimum one litre alcohol is required for each step. Two changes of each grade of alcohol are needed and this change is done after 24 hours. For larger specimen more weeks of time is required. Various reagents can be used instead of ethyl alcohol or along with ethyl alcohol. Some of these are acetone, pyridine, dioxane, butyl alcohol, isopropyl alcohol and cellosolve. After treatment of 100% ethyl alcohol, specimen can be kept in acetone for 24 hours to ensure complete dehydration of specimen.
Clearing - Alcohol is scarcely miscible with paraffin wax and so after dehydration it is necessary to treat tissue with a reagent (clearing agent) that mixes with both substances and which may in turn be eliminated in the process of wax impregnation. For the purpose xylene, cedar wood oil, toluene, benzene, chloroform can be used. We are using xylene as it is colourless and rapid in action. Specimen is kept in xylene for 2 to 8 hours as per size. For larger specimen (more than 200 grams) require 24 hours. Prolonged exposure may make tissue hard and brittle. So colour change in the tissue is noted after each hour. Xylene gives the oily, brown appearance to the tissue.
Paraffin impregnation- In this xylene (clearing agent) is eliminated from the tissue by diffusion into surrounding melted paraffin wax (melting point 52-74) following which wax diffuses into the tissue to replace it. Paraffin embedding bath or incubator is used for paraffin impregnation in which temperature is adjusted 5°C more than melting point of paraffin wax. A jar with melted wax and specimen is kept in incubator. During paraffin impregnation oily brown appearance of tissue starts to disappear and becomes near to natural colour. According the size of tissue paraffin impregnation is done for 4 to 24 hours as per size of tissue. Colouring dye can be added in the paraffin wax to give colour to the tissue.

Finishing - After proper paraffin wax impregnation, specimen is removed from melted wax and just dip for few seconds is given in xylene to remove excess of wax on specimen surface. Now the specimen is allowed to cool at room temperature or lower compartment of refrigerator, so that the wax starts to hard. After complete hardening of wax, specimen takes its natural colour. Colouring of the specimen can be done by using oil based colour to mark important structure. Water based colour can be used before or after the fixation of tissue. Protective coating of the specimen is done with resin like polymer.
After the process of paraffination, water from the formalin fixed specimen is replaced by the paraffin wax. Wax preserves the specimen as in its natural state. Paraffinated specimen is odourless and harmless to handle. Wax models are used for study purpose in medical colleges. Paraffinated specimens are original specimen with wax impregnated so these are better than wax models. By the process of paraffination, specimens of spleen, kidney, brain stem, cerebellum, hemisphere of cerebrum, sections of cerebrum, heart and any part of human or animal body can be paraffinated. The specimens prepared by paraffination should be kept in dry and cold place for longer preservation. Paraffinated specimens are important in educational, research and cultural tools in the medical and veterinary science world.

5. CLAIMS:
1. I claim "PARAFFINATION TECHNIQUE FOR PREPARATION OF MUSEUM SPECIMEN BY IMPREGNATION OF PARAFFIN WAX IN TISSUE" comprising steps i) specimen fixation, ii) dehydration, iii) clearing, IV) paraffin impregnation and V) finishing of which i) specimen fixation is done by immersing biological tissue/organ in 10-40% formalin solution for 10-15 days, later ii) dehydration of tissue is done by immersing it 48 hours in each ascending grade i.e. 70, 90,100% of ethyl alcohol, then iii) clearing is done by immersing dehydrated tissue in xylene for 2-24 hours as per size of tissue and then IV) paraffin impregnation is done by immersing tissue in melted paraffin wax (melting point 52-74), kept in incubator of which temperature is adjusted at 5°C more than melting point of paraffin wax for 4-24 hours and finally V) finishing is done by removing the tissue from melted wax and just dip for few seconds is given in xylene to remove excess of wax on specimen surface and now the specimen is allowed to cool at room temperature or lower compartment of refrigerator, so that the wax in the tissue, starts to hard and later on for protection, coating of resin like polymer is done.
2. I claim step i) fixation of tissue in claim 1 which can be done by any one or combination of fixative solutions like Mercuric chloride- formalin, formal-saline, Susa fixative, Zenker's fluid, Bouins's fluid, Kaiserling solution formed by using any one or combination of fixative agent formaldehyde, glutaraldehyde, mercuric chloride, potassium dichromate, chromic acid, picric acid, osmic acid or acetic acid which causes coagulation of tissue proteins and constituents thus minimise their loss or diffusion during processing.

3. I claim step ii) dehydration of tissue in claim 1 which can be done by any one of the reagent i.e. ethyl alcohol, acetone, pyridine, dioxane, butyl alcohol, isopropyl alcohol and cellosolve or by using two or more reagents which replaces water in tissue and make them dehydrated.
4. I claim step iii) clearing of tissue in claim 1 which can be done by any one of the reagent i.e. xylene, cedar wood oil, toluene, benzene, chloroform or by using two or more clearing reagents which replaces dehydrating reagent in tissue.
5. I claim step IV) paraffin impregnation of tissue in claim 1 which can be done by any type of paraffin wax having melting point from 52-74°C and by using other polymers like cellulose nitrate and synthetic resin which replace clearing agent.
6. I claim process of paraffination in claim 1 which can be carried out without step i) fixation or iii) clearing or both i) fixation & iii) clearing.
7. I claim process of paraffination in claim 1 in which colouring of tissue/specimen can be done by using water soluble dye before or after the fixation of tissue or by using oil soluble dye after step of clearing or step of paraffin impregnation.
8. I claim process of paraffination in claim 1 in which protective coating in step V) finishing can be done by xylene balsam, colponium-terpentine, euparal, DPX or resin like polymer.

9. I claim process of paraffination in claim 1 in which step ii) dehydration can be carried out under refrigeration and step IV) paraffin impregnation can be done under vacuum.