In this article we will discuss about the six important laboratory reagents required in preparation of permanent slides. The laboratory reagents are: 1. Fixing and Killing Agents 90% Alcohol 2. Stains 3. Alcoholic Grades 4. Physiological Salines 5. Mayer’s Albumens 6. Preservatives.
1. Fixing and Killing Agents 90% Alcohol:
70% alcohol: 74 mL.
Distilled water: 26 mL.
Acetic acid:
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Acetic acid glacial: 1 mL.
Distilled water: 99 mL.
Bouin’s fluid (aqueous):
Picric acid
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(Saturated aqueous solution): 75 mL.
Acetic acid glacial 5 mL.
Formaldehyde: 25 mL.
(For Washing Bouin’s fluid-use water or 50% alcohol. For final washing add a few grains of lithium carbonate).
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Bouin’s fluid (alcoholic):
Picric acid: 75 mL.
(Saturated solution in 70% alcohol)
Acetic acid: 5 mL.
Formaldehyde: 25 mL.
(Fixing time: 24 hrs. or longer Washing fluid: 50% alcohol).
Mercuric chloride (Corrosive acid)
Mercuric chloride: 65 mL.
(Saturated aqueous solution)
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Acetic acid: 5 mL.
90% alcohol: 30 mL.
(Washing fluid: 50% alcohol with a few crystals of Iodine).
Buffered Formalin:
10% formalin: 1000 mL.
Sod. di-hydrogen phosphate: 4.00 gm.
Anhydrous disodium phosphate: 6.5 mL.
(For Washing Bouin’s fluid, use water or 50% alcohol. For final washing add a few grains of lithium carbonate).
Zenker’s fixative:
Potassium dichromate: 2.5 gm.
Mercuric chloride: 4.5 gm.
Distilled water: 100 mL
Add Glacial acetic acid 5.00 mL just before use.
(Excellent general fixative). Washing fluid water and water plus Iodine. Fixing time 4-12 hrs.)
Note:
Wherever HgCl2 (Mercuric chloride) is used in fixation; Iodine washing (Iodine-1 gm., Pot. Iodine-2 gm., water-100 cc.) is necessary. Post treatment with Sodium thiosulphate (5% aq. sol.) is also essential in such cases to remove Iodine.
Helly’s fixative
Same as Zenker’s fixative except that here acetic acid is replaced by formalin before use.
Carnoy’s fixative
Chloroform: 30 ml
Acetic acid (glacial): 30 ml
Absolute alcohol: 60 ml
Excellent for Carbohydrates, Glycogen, Nissel substance only; washing fluid: Absolute ale, washing time: 2 to 3 hrs.; Fixing time: 3 to 6 hrs.
Dafano’s fixative:
Cobalt nitrate: 1 gm.
Formalin (40%): 15 ml
Distilled water: 100 mL
(Washing fluid: water; fixing time 12 to 14 hrs.; Good for Golgi apparatus)
2. Stains:
Eosin:
Eosin-G: 1gm
Alcohol 70%: 1000 ml
Acetic acid glacial: 5 ml
Borax Carmin:
Borax: 4 gm.
Distilled water: 100 ml
Add
Carmine: 3 gm.
Heat gently and add
Alcohol 70%: 100 mL
Allow to cool and then filter
Delafieid’s Haematoxylin:
Haematoxylin: 4 gm.
Absolute alcohol: 25 mL
Saturated solutions of Ammonium alum aqueous: 400 mL
Leave open in light and oxygen for 3 days and add
Glycerine: 100 cc.
Methyl alcohol: 100 cc.
Leave for a week to saturate and filter.
Acetocarmine:
Glacial acetic acid: 45 mL
Distilled water: 55 mL
Carmine powder: 0.5-1.0 gm.
Mix acetic acid and water. Heat to boiling and add carmine powder. Shake, cool and filter. Add a crystal of FeCl3.
Leishman’s Stain:
Leishman’s Stain: 0.15 gm.
Absolute alcohol: 100 mL
The stain is used to stain Malarial parasite and other protozoans in blood smears.
Mallory’s triple stain:
Sol. A:
Acid fuchsin: 1 gm.
Distilled water: 100 mL
Sol. B:
Phosphomolybdic acid: 1 gm.
Distilled water: 100 mL
Sol. C:
Anilin blue w.s:. 0.5 gm.
Orange G: 2.00 gm.
Distilled water: 100 mL
(Staining time in sol. A-30 second; in sol. B-3 mts and in sol. C-2 mts; After every solution rinse in distilled water; Directly take the slide to 90% ale. (Very good for connective tissues)
Picro Carmine:
To prepare Picro-carmine take saturated aqueous solution of Picric acid and add Carmine (saturated in NH2OH and dry it until picric acid is saturated with carmine. Filter and use. If it is to be stored for longer time then evaporate to dryness on a water bath till powder is formed.
Picro carmine powder: 1.0 gm.
Distilled water: 100 mL
Picro-indigo Carmine:
Picric acid: 100 mL
Indigo carmine: 2 to 3 gm.
3. Alcoholic Grades:
Different grades of alcohols are prepared for the process of dehydration in permanent slide preparation.
30% alcohol
Distilled water: 65 mL
Alcohol 95%: 35 mL
50% alcohol
Distilled water: 45 mL
Alcohol 95%: 55 mL
70% alcohol
Distilled water: 25 mL
alcohol 95%: 75 mL
90% alcohol
Distilled water: 10 mL
Alcohol 95%: 95 mL
4. Physiological Salines:
Sodium chloride 0.75% for birds tissues
Sodium chloride 0.9% for mammalian tissues except blood.
Sodium chloride 0.8% for Salamanders and other amphibian tissues
Sodium chloride 0.64% for frog’s tissues
Sodium chloride 0.60% for invertebrates.
5. Mayer’s Albumens:
White of egg (Albumen): 50 ml
Glycerin: 50 ml
Sodium salicylate: 1.0 gm.
Shake them well together and filter into a clean bottle for use. It is used to fix the small organisms and microtome sections on the slide.
6. Preservatives:
Preservatives are chemicals capable of preventing microbial infection and thereby stop putrefaction or rotting of animal and plant body.
i. Formaldehyde Solution:
Formaldehyde is a volatile solution. The maximum concentration that can be manufactured is 40%. In preservation, commercial grade of formaldehyde is used. Strength of the solution is calculated on the basis of 40 ml reagent in 100 ml formaldehyde available in the market.
To prepare a 1% solution add 2.5 ml formaldehyde to 50 ml distilled water. Make up volume to 100 ml by adding distilled water. Museum specimens are usually preservated in 4 to 6% solution. For permanent preservation the solution should be changed after 4 to 5 days of first preservation. No further change is required.
ii. Spirit Solution:
70% rectified spirit or 90% ethyl alcohol is a very good preservative. For prolonged preservation the solution should be changed after 4 to 5 days of the initial preservation. No further change is required. In prolonged preservation, however, the tissues become brittle.
iii. Glyceroalcohol:
Glyceroalcohol is an excellent preservative for soft bodied organisms. Not only the soft texture of the muscles are retained but the colour of the organisms too remains unchanged for quite a long time. It is of common use for preservation of specimens to be dissected later. To prepare glyceroalcohol, absolute glycerine (glycerol) and rectified spirit (90% ethyl alcohol) are mixed in 1:1 ratio.