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Ltnit 8 Fixation and Staining Techniques

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Ltnit 8 Fixation and Staining Techniques LTNIT 8 FIXATION AND STAINING TECHNIQUES Structure 8.1 Introduction Objectives 8.2 Introduction to Steps Involved in Permanent Mounts of Plants and Animals Tissue Processing Processing Whole Mounts 8.3 Fixatives and Their Action 8.4 Primary Fixative Groups Coagulant Fixative Non-Coagulant Fixative 8.5 Composite Fixatives Plant Fixatives Animal Fixatives Fixatives and Safety 8.6 Alcohol Series Use of Alcohol Series in Slide Preparation Basis for Procedure of Staining of Permanent Mount 8.7 Some Staining Theory and Methods Basic Dyes Acid Dyes Amphoteric Dyes Types of Staining Methods Mordanting 8.8 Formulary of Reagents and Stains Fixatives in Common Use Stains in Common Use Grades of Alcohol Animal Ringer Solution Animal Physiological Saline Solution 8.9 Summary 8.1 0 Terminal Questions 8.1 1 Answers 8.1 INTRODUCTION - - - - --- - - - - -- - - - - In the previous unit you have already studied about basic techniques of slide preparations. You learnt how slides should be cleaned, cared for, labelled and stored. You also learnt the preparation as well as staining of temporary mounts of plant and animal tissues using squash technique (root-tip) and smear techniques (human cheek cells). The present unit deals briefly with the steps involved in preparing permanent slides of tissues and whole mounts of plants and animals. This unit however deals mainly with the various chemicals that are used for fixing and staining of animal and plant tissues for both temporary and permanent mounts. So in this unit you will study the theory, nature, preparation as well as storage of fixatives, alcohol series, mordants and stains that are used in the preparation of whole mounts or tissue mounts of plants and animals. You will also learn that some fixatives and stains may be used for both temporary as well as permanent mounts, while some may be used exclusively for either temporary or permanent mounts. Microscopy, Staining and While going through this unit you may wonder why you are being taught about Culture Techniques the steps involved in preparing permanent (whole and tissue) mounts of plants and animals as well as the preparation of alcohol series, fixatives and stains that are used exclusively for preljaring such permanent slides or mounts since the preparation of such mounts is not a part of this certificate course. The need for you to learn how to prepare these chemicals is that in all probability you will be asked to prepare them for the various practical exercises being conducted by teaching and research faculty. Objectives After going through this unit you will be able to: briefly list the steps involved in preparing permanent tissues and whole mounts of plant and animal, explain what a fixative is and describe its mode of action, explain the importance of the alcohol series and their mode of action in preparation of permanent mounts, describe the theory for the basis of various stains termed as acid, basic, arnphoteric stain and their mode of action, describe progressive and regressive staining methods, define mordants and explain their use in permanent slide preparations, and prepare the commonly used fixatives, alcohol series and stains and mordants that are used in plant and/or animal tissue preparations. 8.2 INTRODUCTION TO STEPS INVOLVED IN PERMANENT MOUNTS OF PLANTS AND ANIMALS In order to keep the slide of whole mount or tissue mount of plant or animal for long term reference in terms of days and even years, it must be made as a permanent preparation. This is achieved for (1) tissue and (2) whole mount by the following steps. 8.2.1 Tissue Processing 1. Fixation and preservation of the tissue material. 2. Dehydrating the material with increasing concentrations of alcohol (30%, 50%, 70%, 90%, loo%, absolute alcohol) in order to prepare it for infilteration with paraffin wax. 3. Section cutting of tissue by microtome. 4. Clearing the wax infilterated section with a solvent like chloroform or xylene that is mutually soluble with alcohol and wax and which causes minimal hardening and shrinkage. 5. Hydrating the tissue for preparing it to take up stain, by treating it with decreasing concentrations of alcohol - loo%, 95%, 90%, 80%, 70%, 50%, 30%, distilled water. 6. Staining with required stain. 7. Dehydrating the tissue again as before, for mounting by using increasing concentrations of alcohol - 30%, 50%, 70%, 90%, 100% (absolute alcohol). 8. Clearing the tissue once again with a solvent like clove oil or xylene which is mutually soluble with alcohol and the mounting medium . .- - 9. Mounting the tissue on a slide, under a coverslip in a preservative like Fixation and Staining Canada balsam or Euparol or DPX which dries hard and has a refractive Techniques index, similar to glass. 8.2.2 Processing whole mounts The reagents and stains are similar but the process is shorter and involves the following steps: i) Fixation or preservation of whole mount. ii) Staining with required stain. iii) Dehydrating the mount by using increasing strengths of alcohol. iv) Clearing the tissue with clove oil or xylene which is a solvent that is mutually soluble with alcohol and the mounting media Canada balsam or Euparol. v) Mounting the whole animal or plant on a slide under a coverslip in a mounting media like Canada balsam or Euparol or DPX which is a preservative. 8.3 FIXATIVES AND THEIR ACTION Tissue is prepared for inicroscopic study in various ways. You are aware from Unit 7 of this course that if the mount you make is to be anything but very temporal;, the tissue within the mount must be preserved in some way. Fixation is a special case of preservation, special in that the biologist aims to use specific fixatives and related stains that tend to preserve and stain those tissue structures which he/she plans to study. You should also be aware that to make permanent microscopical mounts, the sections that you cut must be dehydrated in order to be made compatible with mounting media (like Canada balsam) which can be dissolved in solvents such as xylene. Xylene is a very efficient solvent for fats. Tissue which has been newly removed from the plant or animal and has not been preserved in any way is called fresh and may be justifiably said to be in the living state. Fresh tissue has all of its cellular structures (tissue elements) present, and if it were to stay that way indefinitely you would not be reading this course! It can stay in this state for a short time if kept in physiological saline or Ringer's Solution. However, it is a fact that fresh tissue does not remain in its fresh state for very long despite saline or Ringer's solution, because in the cell there are membranes, mitochondria, Golgi apparatus and a host of other structures which the cell's own enzymes start to break down as soon as the tissue is excised from its source. The process is called autolysis, and soon renders the tissue so unlike its living slate as to make it useless for detailed examination. The first purpose of fixation then is to prevent the tissue from breaking down by its own enzyme systems. It may therefore also be said with some justification that the fresh tissue, by being fixed, is also killed, indeed small protistans and not so small metazoans, to say nothing of plants, are often killed by immersion in, or treatment with fixatives. If slides were to be prey to bacteria and other agents of decay we should not expect them to last for long in any usable stage. Many of the chemical agents we use as preservatives, simply to prevent the microbiological decay of tissue, are also used as fixatives, or are mixed with fixatives to produce a fluid which will ensure the attainment of both aims. Microscopy, Staining and Above and beyond the fulfilment of these purposes, we further expect to be Culture Techniques able to colour or stain the material on which we are working, so that we can actually see the different tissue elements we have taken such great pains to preserve. This implies some preparative function of the fixative, and it is indeed well known that certain fixatives are conducive to good results with certain stains while others are not. It is also known that certain fixatives will preserve certain tissue elements with greater success than others, which means that a fixative can be 'designed' to do a specific job, i.e. to pick out, when used in combination with the appropriate stain, a particular tissue element. To sum up, we can therefore say that the general effect aimed for during fixation is that of rendering the tissue in a state which is as close as possible to the living state (i.e. with minimum distortion). It would be of little use to look at slides which bear no resemblance to the living material, so clearly we must ensure that each tissue element is at least visible in the finished mount, or, if it is not visible, we should know the reason as to why it cannot be seen. Fixatives then, are, chemical agents which: (1) preserve tissues in as life-like a manner as possible for microscopical examination, with each required tissue element at least represented; and (2) prepare them for subsequent processing so they are receptive to the appropriate stains and so that the required elements will not be dissolved from them by such processing. Since we have already said that fixatives are also preservatives, we expect that the tissues will remain in that state for a long period of time. SAQ 1 If the first aim of a fixative is to prevent the autolysis (self break down) of tissue by its own enzyme systems, what do you think might be the second aim? 8.4 PRIMARY FIXATIVE GROUPS Fixatives are generally classified into two primary groups by the effect that they have on the protein constituents of cells.
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