In the laboratory study of biological tissues and other materials, various types of chemicals are needed for specific results. Such chemicals are mainly the preservatives, stains and some other reagents used in various experiments. Some such chemicals are being explained in this lesson.

After completing this lesson you will be able to :

• differentiate between reagents, stains and preservative.
• list the commonly used preservatives, stains and reagents.
• state the chemical composition ofBouin 's fluid, Carnoy mixture, Leishman 's stain, Safranin, Acetocarmine, Methylene blue. Iodine solution, Benedict's solution, Fehling's solution. Ringer's solution, FAA (Formalin Acetic Acid-Alcohol) solution.
• describe the uses of above mentioned solutions, stains

Let us first define the above three terms to know the difference between them.

These chemicals are used to kill and fix plant/animal tissues and specimens in such a way that they retain their original shape, form and size. These make the tissues hard and prevent them from decaying. Some of the preservatives are as follows :

(1) Bouin's fluid: This preservative or fixative penetrates rapidly in the tissues, for making histological preparation. Composition:

Saturated aqueous picric acid - 75 ml

Formalin (40% Formaldehyde) - 25 ml

Glacial Acetic Acid - 5 ml

(2) Carnoy's fluid (mixture): It penetrates rapidly and gives excellent nuclear fixation.

Composition

Absolute Alcohol - 60 ml

Chloroform - 30 ml

Glacial Acetic Acid - 10 ml

It is prepared fresh. Care is to be taken as it is highly poisonous and inflammable.

This is very good fixative and tissues may be left for a long period without harm.

Composition

50% Alcohol - 100 ml

40% Formaldehyde - 6.5 ml

Glacial Acetic Acid - 2.5ml

There are different stains which are used in the preparation and study of different biological materials. Staining can be done in three ways.

1. Single Staining : Where only one stain is used giving a single colour to ihe tissue

2. Double Staining : Where two stains are used, each stains a specific area or the particular cell organelle.

3. Multiple Stianing: More than two stains are used in the preparation of slide of tissue or organelle. Each stain will colour only the specific organelle of the cell. Some of the stains are dealt below :

(1) Leishman's Stain : It is a readymade double stain, used for staining blood films. It

gives blue colour to the nucleus and pink to the cytoplasm.

Leishman stain powder - 15gm

Methyl alcohol (solvent) - 100 ml

For good result, this stain is kept in dark coloured bottle.

(2) Safranin : It is used as a general stain for plant tissues. The stain may be prepared both

in water as well as in 90% alcohol depending on the requirement.

Safranin powder - 1 gm

Distilled water - 100ml

It is a synthetic dye which gives pink or red colour to the object stained.

(3) Acetocarmine : It is mainly used to stain chromosomes in the study of cells.

Glacial acetic acid - 45 ml

Carmine powder - 2 gm

Distilled water - 55 ml

(4) Methy kne blue: This stain may be used both as aqueous or alcoholic. It is a basic stain

and mainly stains nucleus and fungal bodies.

Aqueous Methylene blue :

Methylene blue - 100 mg

Distilled water - 100ml

The stain is dissolved in distilled water Alcoholic Methylene blue:

Methylene blue - 0.3 gm

95% Ethyl alcohol - 30 ml

Distilled water - 100 ml

This stain is prepared by adding 30 ml of saturated alcoholic solution of methylene blue (0.3 gm of it to 30 ml of 95% ethyl alcohol) in 100 ml of distilled water.

There are different reagents which are used to test the different substances present in certain solutions. Some of them are given below which are commonly used in a biology laboratory.

(1) Benedict's Solution : It is used for the test of sugar.

Copper sulphate - 1.7 gm

Sodium citrate - 17.3 gm

Sodium carbonate (anhydrous) - 10.0 ml

Distilled water - 1000 ml

Dissolve 17.3 gm sodium citrate and 10 g of anhydrous sodium carbonate in 600 ml of distilled water. Filter the solution. Simultaneously prepare copper sulphate solution. Add this solution slowly to the previous filtered solution constantly stirring it. Add enough distilled water to make a total of 1 litre.

(2) Fehling's Solution A and B : It is also used for testing of sugar. It is commonly

purchased ready made from the market.

Copper sulphate - 34.6 gm

Distilled water - 500 ml

Sodium hydroxide - 175 gm

Sodium potassium tartarate - 173 gm

Distilled water - 500 ml

When testing for sugar equal amounts of Fehling's solution A and Pehling's solution B are

added to the solution which is to be tested.

(3) Iodine Solution : It is commonly used for testing starch. As such it is brownish in

colour.

Iodine - 0.3 gm

Potassium iodide - 1.5 gm

Distilled water - 100 ml

This solution is isotonic to that of tissues. It does not spoil quickly and living material can

be placed in it for observation in normal living state.

Potassium chloride - 0.42 gm

Sodium chloride - 9.0 gm

Calcium chloride - 0.24 gm

Sodium bicarbonate - 0.20 gm

• Preservative is a substance or method which prevents decay and decomposition of organisms or their parts.
• Stain is a chemical which colours tissue for study.
• Chemical is a substance obtained by a chemical process or used for producing chemical effect.
• Different type of preservatives are used for different objects and for different purposes.
• The various types of stains are used for various tissues or animals.
• Staining may be single, double or multiple
• Different type of reagents are used for different experiments.

1. Define the term Reagent

2. What is meant by (i) Double staining and (ii) Multiple staining?

3. Mention the use and the composition of Bouin's fluid.

4. Mention the components of F.A.A.

5. Which cell organelle is readily stained by Leishman's stain?

6. Name one stain used generally in biology laboratories.

7. Give the composition of Fehling's Solution A and B. Mention the substrate it is used for testing.

8. Mention the use of Ringer's solution.

1. As given in Section 40.3

2. Refer to Section 40.4

3. Penetrates rapidly in tissues

4. Water/CO₂/C₂H₅OH