Fixation and Tissue Processing Anatomic Dissection

Question: Describe about the Fixation and Tissue Processing for Anatomic Dissection. Answer: Introduction The preparation of tissues for experimental purpose involves the anatomic dissection of rats. The tissues taken from the rat are of trachea, oesophagus, lung, duodenum, liver, kidney and femur. The tissues obtained from the dissection are separated from the rat and are subjected to irrigation by normal saline after the division into smaller pieces for the experiment. Then the fixation of the tissues is done by keeping the tissues in 10% Normal Buffered Formalin (NBF), (1) which also helps in preserving the cells and tissues and maintains the viability of the cells and tissues. The tissue processor machine fixes the tissues and then performs the processing. The steps involved in processing are as following; the tissues are dehydrated by increasing the alcohol concentration. Care should be taken tom prevent the tissues from damage. Therefore the concentration of alcohol is raised slowly and effectively. To remove the alcohol from the tissues and prevent the alcohol from reaching in the tissues, Xylene agent is used. The Femur tissues mainly consist of calcium salt and to prepare the tissues for the processing it is important to decalcify the tissue. As the completion stage is attained for the tissues, it needs to be embedded in right proportion of the cassette to perform the sectioning. The tissues in the histological stage need to be placed properly in the cassette. The sectioning of the tissues is performed after the solidification of the blocks. The aim of sectioning is to obtain slice of tissues having a thickness similar to one cell. The microtome used for the sectioning should be placed at 10m, and the blocks should be snipped for obtaining the clear view of the given specimen. Then the microtome thickness should be adjusted to 4m, as it is the standard measurement for daily diagnosis. After the block preparation the tissues are put in the water bath having a temperature about 37 degrees Celsius. This helps removing the rigidity of the folds in the specimen before placing them on the microscopic slide. Haematoxylin (2) is used to stain the acidic nucleus in the specimen. Eosin is used to stain the muscles and collagen having basic properties. The HE method is preferred to get the differentiated tissue sections and to facilitate the morphological study of the tissues. Special stains are required for the investigation and for the diagnosis of liver, duodenum, oesophagus, trachea kidney. The special stains used to perform the experiment are Periodic Acid Schiffs Diastase and Periodic Acid Schiffs. (3). Write a processing schedule for an urgent endoscopic biopsy eg. Duodenum? Chemical reagents Vacuum Temperature (C) Time Formalin Use Forty Fifteen minutes 70% alcohol Use Forty Fifteen minutes 80% alcohol Use Forty Fifteen minutes 90% alcohol Use Forty Fifteen minutes 95% alcohol Use Forty Fifteen minutes Absolute alcohol Use Forty Fifteen minutes Absolute alcohol Use Forty Fifteen minutes Absolute alcohol Use Forty Fifteen minutes Xylene Use Forty Fifteen minutes Xylene Use Forty Fifteen minutes Xylene Use Forty Fifteen minutes Wax Use Forty Fifteen minutes Wax Use Forty Thirteen minutes Wax Use Forty Thirteen minutes Please list the different types of fixation available? Aldehydes Alcohols Oxidizing agents Picrates Mercurials What are the other clearing agents that could have been used for processing your specimens? Briefly discuss their advantages and disadvantages. Clearing agents Advantage Disadvantage Chloroform Safe for stissue. Need to catalyst Harm and toxic Limonene-derivatives Unharmed Less toxic Stink Cedarwood Oil Acting Slowly High cost Inflammable What precautions should be taken when setting and loading a tissue processor? The precautionary measures taken before setting and loading a tissue processor are The tissues should be fix appropriately The environment of the experiment should be set according to the guidelines maintaining the correct time and temperature settings. The decalcification of the bones should be done properly. (4). What effects can have prolonged decalcification have on tissues? The extended decalcification affects the histological characteristics of the tissues. Excessive decalcification can influence the quality of the stain and this would affect the clarity in observation. What methods other than a chemical test for calcium ions are available for determining the end point of decalcification? Comment on their reliability. To determine the end point of decalcification of the tissues some other method which could be applied can be X-rays- it is authentic and commonly used method. The other method could be manual methods but they are not reliable and can cause harmful effects on the tissue. (5). When embedding tissues, what should be avoided in order to ensure that quality is achieved? To ensure that the quality is achieved following should be considered. Do not use a cassette which is improper. The forceps should not be over heated while handling the tissues. The mould should not be filled excessively The size of the mould should not be similar for every specimen Take care of the force while handling the specimen. Excess force can spoil the specimen. (6). References Kiernan JA. Fixation and Tissue Processing. Special Stains and HE. Available from www.dako.com/08066_12may10_webchapter16.pdf [accessed 10 October 2016]. Fehlert et al. Revised guides for organ sampling and trimming in rats and mice- part 1. Exp toxic pathol. 2003; 55: 91-106. Gallik S. Tissue processing histological stains. Histology OLM. Available from https://histologyolm.stevegallik.org/node/35. 2009. [accessed on 10 October 2016. Rolls G. An introduction to Decalcification. Leica biosystem. Available from https://www.leicabiosystems.com/pathologyleaders/an-introduction-to-decalcification/ [Accessed on 10 October 2016]. An YH, Martin KL. Bone sectioning. Handbook of histology methods for bone and cartilage. USA: Springer science business media. 2003. Krause WJ. Getting started. The art of examining and interpreting histologic preparations: A laboratory manual and study guide for histology. USA: Universal publisher; 2004.