PREPARATION OF SPECIMENS FOR HISTOLOGIC STUDY
1. PREPARATION OF SECTIONS OF PARAFFIN EMBEDDED SPECIMENS 2. PREPARATION OF SECTIONS OF PARLODION EMBEDDED SPECIMENS 3. PREPARATION OF GROUND SECTIONS OF TEETH
OR BONE
4. PREPARATION OF FROZEN SECTIONS
5. TYPES OF MICROSCOPY
1. PREPARATION OF SECTIONS OF PARAFFIN EMBEDDED SPECIMENS
1.1. Obtaining the specimen : Specimen taken from humans or experimental animals must be removed without crushing either while the animal is alive or immediately after it has been killed.
Fixation of specimen : Most commonly used fixatives for dental tissues is 10% neutral formalin.
1.2.
Purpose of fixation are to coagulate the protein thus reducing alteration by subsequent treatment.
Dehydration of the specimen : Specimen is gradually dehydrated by being passed through a series of increasing percentages of alcohol (40%, 60%, 80%, 90% and absolute alcohol, remaining in each dish for several hours.
1.3.
• Since paraffin and alcohol are not miscible, the specimen is passed from alcohol through two changes of xylene.
Infiltration of the specimen with prarffin :
1.4.
•Specimen is infiltrated with paraffin by placing it in a dish of melted embedding paraffin and the dish is put into a constant tempreture oven regulated to above 60 o C.
• Time in the oven depends upon the size and the density of the specimen.
1.5. Embedding the specimen :
•Specimen is embedded in the centrer of a block of paraffin, a small paper box, perhaps a 19 mm cube for a small specimen is field with melted paraffin and the specimen is placed in the centre of the box of paraffin.
• The mounted paraffin block is trimmed with a razor blade so that there is about 3 mm of paraffin surrounding the specimen on all four sides.
1.6. Cutting the sections of specimen :
•Microtome is adjusted to cut sections of the desired thickness (usually 4 to 10 um.).
1.7. Mounting the cut sections on slides :
•The preparation of the slides is done by coating of clean slides with a think film of Meyer’s Albumin adhesive.
• A prepared slide is slipped under the paraffin ribbon and then is lifted from the water with the ribbon which content tissue sections, arranged on its upper surface. • The slide is a placed on a constant tempreture of about 40 C.
1.8. Staining the sections:
•One combination of stains mostly used for routine microscopic study is hematoxylin and eosin commonly known as H & E.
• The dried slides are placed vertically in glass staininng trays the trays are passed through a series of staining dishes that contain the various reagents.
2. PREPARATION OF SECTIONS OF PARLODION EMBEDDED SPECIMENS
Obtaining the specimen : •The specimen is separated as carefully as possible by means of a sharp scalpel and a bone saw.
2.1.
2.2. Fixation of the specimen :
•For fixation specimen is immediately placed in about 400 ml of 10% neutral formalin
2.3. Decalcification of the specimen :
•It is done by suspending the specimen in about 400 ml of 5% nitric acid. Acid is changed daily for 8 to 10 days. •To test for complete decalcification a needle is pierce to the hard tissue. When it enters bone and tooth easily, then it is ready for further treatment.
• Another way to test for complete decalcification. Another test is done by placing the specimen in the test tube containing 5 – 6 ml of acid and then adding 1 ml of concentrated ammonium hydroxide in several drop saturated aqueous solution of ammonium oxide
• If no precipitate is detected after the test tube as stood for an hr and after several addition of ammonium oxalate, It may be assumed that specimen is almost completely decalcified
2.4. Washing the specimen:
•The specimen is washed in running water for at least 24 hours to remove all acid.
2.5. Dehydration of the specimen :
Dehydration of the specimen : Specimen is gradually dehydrated by being passed through a series of increasing percentages of alcohol (40%, 60%, 80%, 90% and absolute alcohol, remaining in each dish for several hours.
• It should be placed in several changes of absolute alcohol over a period of 48 to 72 hours.
2.6. Infilteration of the specimen with parlodion: •The specimen is transfer to 2% parlodion covered tightly to prevent evaporation and allowed to stand for a period of from 2 weeks to a month.
• From 2% parlodion the specimen is transferred to increasing percentages of parlodion (4%, 6% , 10% and 12%)
2.7. Embedding the specimen in parlodion :
•The specimen is embedded in center block of parlodion. •12% parlodion is poured into the glass dish with straight sidewalls and a lid and then specimen is placed in parlodion.
• Then more parlodion is added in glass dish so that there is 13 mm of parlodion above the specimen. • This process required two to three weeks.
2.8. Cutting the section of specimens :
•Sections are cut with sharp microtone knife and then place in to a flat dish of 70% alcohol, so that it do not became dry.
2.9. Staining the sections:
•The sections are passed through the serious of reagents separately or in groups of three or four, using a perforated section lifter to make the transfer.
3. PREPARATION OF GROUND SECTIONS OF TEETH OR BONE
Extracted teeth should be preserved in 10% formalin until used.
3.1.
3.2. The tooth cut longitudinally in a mesiodistal plane. Then it is held securely in the fingers and its buccal surface is applied firmly to that flat surface of the rapidly rotating wheel. The cut surface of the tooth is ground again by using fine abrasive lathe wheel until the level of desired section is reached
3.3. The wooden block is applied with adhesive tape in a way sach that, sticky side of the tape is diercted outward. The ground surface of tooth is pressed onto the adhesive tape on one side of the wooden block. The lingual surface of the tooth is applied to the coarse abrasive lathe wheel. And the tooth is ground down to a thickness of about 0.5 mm.
3.4. A drop of mounting medium is placed on the slide and the section is placed on the drop, another drop of mounting medium is put on the top of the section and cover glass is affixed of the microscopic studies.
4. PREPARATION OF FROZEN SECTIONS •Fixed soft tissues or fresh unfixed soft tissues may be cut in to the sections 10 to 15 micro meter. By freezing the block of tissue with either liquid or solid carbon dioxide and cutting it on a freezing microtome.
• Frozen sections can be quickly prepared and useful if immediate examination of specimen is required.
5. TYPES OF MICROSCOPY
•Many types of microscope are used for study of tissues. Most common is the bright field microscope.
• Modification of the instrument have provided the phase contrast interference, dark field, and polarizing microscope.
SUBMITTED BY :
SURAJ MASATKAR
(INTERN 2004)