Dissolve, dilute, definitions
Making a solution[4]
1. A solution consists of:
- the dissolved substance
- the solvent
2. After dissolution, the quantity of dissolved substance is in a given quantity of solution: i.e. not in the solvent. Example: y ml should be in 10 y ml solution. So not in 10 y ml of solvent; because then there would eventually be 10 y + y ml of solution.
3. Dissolve the quantity of substance to be dissolved in less than the required amount of solvent and then make up to the required volume. Example:
200 ml of 3% sugar solution to be made.
- Calculate how many grams of sugar is needed (6 grams);
- dissolve 6 grams of sugar in 100 ml of water;
- fill up to 200 ml water.
So do not: take 200 ml water and dissolve 6 grams of sugar in it.
4. The generally valid equation is: number of grams of substance to be dissolved = % by weight of solution X number of ml of solvent / 100
Making a dilution[4]
1. Calculation:
This is based on a pre-existing solution, of which another solution is made.
a. Example:
1.5 litres of 8% kitchen salt solution to be made.
- In stock is a solution of 40%
- In 1 liter stock solution of 40% there is 400 gr cooking salt;
- to make 1.5 liters of 8%. So it contains 80 gr cooking salt per litre, or 120 gr per 1.5 litres;
- of the stock is therefore necessary:
- 120/400 X 1 litre = 0.3 litre;
- 0.3 litres of the stock is now supplemented with 1.2 litres of water to 1.5 litres of solution of 8%.
b. is the general rule:
- determine the concentration of the required solution;
- add to the number of ml which also indicates the concentration of the requested solution such quantity of solvent as will give the concentration of the initial solution.
2. Table for making dilutions
Application table:
Available | Ethanol | 95% |
Required | Ethanol | 70% |
| Take | 70ml | 95% |
| Add | 25ml | aqua dest. |
Acquired | 95ml | 70% |
Making a dilution series[4]
1. By diluting a solution, re-diluting the diluted solution and going on like this, one obtains a so-called dilution series, which can achieve extremely low concentrations, which may be necessary for biological experiments. In this way one can also test e.g. a PH meter.
2. The procedure is usually as follows:
a. A number of test tubes are numbered consecutively, starting with 1;
b. 10 ml of a solution of known concentration is placed in the first test tube;
c. 9 ml of solvent (AD) is placed in each of the remaining test tubes;
d. Extract from tube 1 by pipette 1 ml and transfer to tube 2;
e. tube 2 is shaken well;
f. Extract from tube 2 with a clean 1ml pipette and transfer to tube 3;
g. the sequence is continued in this way, so that each time 10x dilutions are made.
Definitions[4]
1. Mol
a. An amount of substance that contains as many molecules as there are atoms in exactly 12gr of the nuclide 12C. The mole has been added to the SI as a basic unit. The Mole is intended to replace the gram molecule.
b. By a mole of a substance we mean a quantity of 6.02 x 1023 atoms, ions or molecules of the substance.
c. A mole of atoms, ions or molecules of a substance has a mass of as many grams as the atomic, ion or molecule mass.
2. Molality
The number of mill dissolved substance per kg solvent.
3. Molarity
The number of mill dissolved substance per litre of solution.
4. Grammolekuul
The quantity of a pure substance of which the weight is as many grams as the molecular weight (= sum of the atomic weights of the individual atoms of the molecule) of that substance is large.
5. Grame equivalent
a. The quantity of the substance that is capable of binding or releasing 1 Mol H+ ions.
b. 1 greg = 1 Mole/skill.
6. Normality
The number of gram equivalents of a substance contained in 1 litre of solution.
7. Molecular weight (M) = relative molecular mass
a. The ratio of the mass of the molecule to the twelfth part of the mass of the 12C isotope.
b. Using the chemical formula of a substance, the molecular weight can be calculated directly by summation of the atomic weights of all the atoms contained in the molecule defined in a similar manner.
The concentration of solutions[4]
1. Molarity
a. A 1 molar solution contains 1 gram molecule of dissolved substance per 1 litre of solution.
b. Example:
The molecular weight of HCL (hydrochloric acid) is 36,5
A 1M HCL solution thus contains 36.5gr HCL per litre of solution.
2. Normality
a. A normal solution contains 1 g of equivalent dissolved substance per litre of solution.
b. Example:
The molecular weight of HCL is 36,5;
a 1 N solution of HCL contains 36,5 g per litre.
The molecular weight of H2SO4 is 98;
a 1 N solution of H2SO4 contains 49 g per litre
Note: A 1 M solution of H2SO4 contains 98 g per litre.
3. Percentage
a. Volume percents: a 1% solution contains 1ml dissolved in 100ml solution.
b. Weight percent: a 1% solution contains 1gr dissolved in 100gr solution.
c. Example: 1 litre of X solution in 3% water contains 30ml of X and 970ml of water.
4. Conversion from molarity to normality
a. Multiply the given molarity by molecular weight/equivalent
b. Example:
0,8 M H2SO4 solution, molecular weight is 98; equivalent is 49
So: 0.8 X 98/49 = 1.6
Normality is 1.6 N
5. Converting normality to molarity
a. Multiply the given normality by its equivalent/molecular weight
b. Example:
0,5 N Na2CO3 solution, molecular weight is 106; equivalent is 53
So: 0.5 X 53/106 = 0.25
The molarity is 0.25 M
6. Molarity and normality of some acids and bases
Reagents | Formula | Molarity | Normality | % |
glacial acetic acid | HC2H3O2 | 17M | 17N | 99,5 |
| diluted glacial acetic acid | 6M | 6N | 34 | |
| concentrated hydrochloric acid | HCL | 12M | 12N | 36 |
| diluted hydrochloric acid | 6M | 6N | 20 | |
| concentrated nitric acid | HNO3 | 16M | 16N | 72 |
| diluted nitric acid | 6M | 6N | 32 | |
| concentrated sulphuric acid | H2SO4 | 18M | 36N | 96 |
| diluted sulphuric acid | 3M | 6N | 25 | |
| conc. ammonium hydroxide | NH4OH | 15M | 15N | 58 |
diluted ammonium hydroxide | 6M | 6N | 23 | |
| conc. sodium hydroxide | NaOH | 6M | 6N | 20 |
Commonly used animal tissue fixatives[2]
1. Bouin
- 15ml picric acid 1.2% (3E-086[3]);
- 5ml formalin 40% (3D-084[3]);
- 1ml glacial acetic acid (3D-087[3]).
Is also ready for sale: (2C-273[3]).
Fixative penetrates well. Small pieces a few hours, larger ones a few days. Possibly half a day warm (35°C) for fixing large pieces. Then fix cold, not in the refrigerator. Longer fixation does not damage the tissue. Most stainings can be done well to very well. After fixation do not rinse in water but immediately in 70-80% ethanol.
2. Carnoy
- 60ml absolute alcohol;
- 30ml chloroform;
- 10ml glacial acetic acid (3D-087[3]).
Fixative penetrates very quickly into tissue. Pieces of 1 to 2 mm are fixed in 1 hour. Larger pieces are fixed after a maximum of 3 hours. Fixate tissue no longer because otherwise it will shrink too much and become too hard. Immediately after fixation, 2 x 24 hours in absolute alcohol (refresh once).
3. Flemming
- 15ml chromic acid 1% (3G-090[3]);
- 4ml osmic acid 2%;
- 1ml glacial acetic acid (3D-087[3]).
Is also ready for sale: (3D-015[3]).
Fixative penetrates slowly and it is recommended to choose thin pieces of tissue (1 - 3 mm). After 24 hours tissue is fixed and rinsed thoroughly with water (24 hours). After this fixation, staining with haematoxylin is no longer possible.
4. Formaline
- 10ml formalin 40% (3D-084[3]);
- 100ml AD.
Fixative penetrates the tissue relatively slowly. Small pieces 1 day, larger pieces 3 - 4 days. Longer fixation does not harm at all. It can even be stored for months (add a small chunk of marble to prevent the formation of formic acid). After fixation, rinse thoroughly in tap water. After formalin, many stainings are possible.
5. Zenker
- 5gr sublimate (mercuric chloride);
- 2.5gr potassium dichromate;
- 1gr sodium sulphate (3G-066[3]);
- 100ml AD;
- just before use, add 5ml glacial acetic acid (3D-087[3]).
Zenkeris also ready for sale: (3D-038[3]).
Thin slices of tissue are fixed after 24 hours. Rinse in tap water for at least 24 hours. In case of sublimate fixation solutions, it is necessary to remove the sublimate precipitate. This can be done at specimen level with iodine iodine alkali and sodium thiosulfate.
Attention! Mercury chloride is a highly toxic, crystalline, white powder. 0.5gr is lethal for humans!
6. Formaline-alcohol
- 10ml formalin 40% (3D-084[3]);
- 20ml ethanol 80%.
Fixation time 1 - 2 days, larger pieces a few days longer. Fixative gives particularly good results for blood-rich tissue because the granulocytes are better preserved than in pure alcohol.
7. Helly
- 5gr sublimate (mercuric chloride);
- 2.5gr potassium dichromate;
- 1gr sodium sulphate (3G-066[3]);
- 100ml AD;
- just before use, add 5ml formalin 40% (3D-084[3]).
Fixation time 1 - 6 hours. Rinse thoroughly with water (24 hours). Granulocytes keep better than with Zenker. It is necessary to remove the sublimate precipitate in case of sublimate fixation solutions. This can be done at preparation level with iodine-iodic alkali and sodium thiosulfate.
Attention! Mercury chloride is a highly toxic, crystalline, white powder. 0.5gr is lethal for humans!
8. Susa according to Heidenhain
- 4.5gr sublimate (mercuric chloride);
- 0.5gr cooking salt;
- 2gr trichloroacetic acid (3L-002[3]);
- 4ml glacial acetic acid (3D-087[3]);
- 20ml formalin 40% (3D-084[3]);
- 80ml AD.
Fixation time 1 - 24 hours. After fixation, transfer immediately to 90% ethanol. After Susa can, the tissue is very stainable. In case of sublimate fixation solutions it is necessary to remove the sublimate precipitate. This can be done at specimen level with iodine iodine alkali and sodium thiosulfate.
Attention! Mercury chloride is a highly toxic, crystalline, white powder. 0.5gr is lethal for humans!
9. Stieve
- 76ml saturated sublimate;
- 20ml formalin 40% (3D-084[3]);
- 4ml glacial acetic acid (3D-087[3]).
Fixative penetrates very quickly so that even larger pieces of tissue can be fixed well. Rinse in alcohol 90%. It is necessary to remove the sublimate precipitate in sublimate fixative solutions. This can be done at specimen level on the front glass with iodine-iodic alkali and sodium thiosulfate.
Attention! Mercury chloride is a highly toxic, crystalline, white powder. 0.5gr is lethal for humans!
10. Regaud
- 80ml potassium dichromate 3% (3L-110[3]);
- 20ml formalin 40% (3D-084[3]).
Fixative does not penetrate quickly. Fixation time 4 days. After that another 8 days nafixate in potassium dichromate 3%. Then wash out in water. Fixative is excellent for mitochondria.
Frequently used solutions for staining[1]
1. Picric acid alcohol (3D-111[3]) (differentiate with e.g. trichrome according to Masson)
- 1 part ethanol 96%;
- 2 parts alcoholic saturated picric acid solution.
2. Phosphorus molybdenic acid (differentiate with e.g. trichrome according to Masson)
- Dissolve 1g phosphorus molybdenic acid (3D-098[3]) in 100ml AD.
3. Phosphorformolybdenic acid (pickling with e.g. AZAN trichrome according to Heidenhain)
- Dissolve 5g phosphorus molybdenic acid (3D-098[3]) in 100ml AD.
4. Ethanol glacial acetic acid (trichrome according to Masson)
- Add 0.1ml glacial acetic acid (3D-087[3]) to 100ml ethanol.
5. Acid alcohol (AZAN trichrome according to Heidenhain)
- Add 1ml glacial acetic acid (3D-087[3]) to 100ml ethanol.
6. Phosphoric molybdenic acid-Orange G (2C-280[3]) (differentiate with e.g. trichrome according to Goldner)
- Dissolve 3.5gr phosphorus molybdenic acid (3D-098[3]) or phosphorus tungsten acid (3D-092[3]) and 2gr Orange G (1B-221[3]) into
100ml AD.
7. Lugol (3D-072[3]) (remove mercury chloride from sample)
- Dissolve 2gr potassium iodide (3L-005[3]) in 5ml AD;
- Add 1gr iodine (3L-004[3]) which will dissolve in a few minutes;
- supplement with AD to 300ml.
8. Sodium thiosulfate (remove mercury chloride from preparation)
- Dissolve 2.5gr sodium thiosulfate (3L-020[3]) in 100ml AD.
9. Aniline alcohol (differentiate with e.g. AZAN according to Heidenhain)
- Add 1ml aniline (3D-093[3]) to 1000ml ethanol 96%.
10. Periodic acid (e.g. PAS reaction)
- Dissolve 1gr periodic acid (3F-123[3]) in 100ml AD.
11. Potassium permanganate (oxidize)
- 0.25gr potassium permanganate (3G-039[3]) in 100ml AD.
12. Oxalic acid (remove brownstone after oxidation with potassium permanganate)
- Dissolve 5gr oxalic acid (3G-068[3]) in 100ml AD.
13. Oxidizing Haematoxylin (Artificial Maturation)
To oxidize 1 gram haematoxylin to Haematin is needed:
- 177mg potassium permanganate (KMnO4) or
- 114mg potassium chlorate (KCLO3) or
- 200mg potassium iodate (KJO3) or
- 197mg sodium iodate (NaJO3) or
- 276mg potassium dichromate (KCr2O7)
14. Decolourisation of excessively coloured haematoxylin stain
- Dissolve 2ml glacial acetic acid in 100ml AD or
- 0.1ml to 0.25ml hydrochloric acid in 100ml AD.
15. Hydrochloric alcohol 3% (differentiate from e.g. iron haematoxylin)
- Mix 3ml hydrochloric acid 25% (3L-012[3]) with 100ml ethanol 70%.
16. 3M Potassium chloride (storage liquid electrode PH meters)
- Potassium chloride has a molar mass of 74,55 gr/mol.
- 3 x 74,55gr = 223,65gr is needed for 1000ml of water.
- For 100ml: weigh 22,4gr and dissolve this in e.g. 80ml AD. Then fill up to 100ml.
17. 1N Hydrochloric acid (needed to make e.g. Schiff's reagent)
- A 1N solution contains 36.453 grams of hydrochloric acid per litre and therefore corresponds approximately to a value of 3.55%.
18. 1N Sodium hydroxide, NaOH (required to produce e.g. formaldehyde from paraformaldehyde)
- NaOH has a Mole mass of 39,997 gr per mole.
- To make a 1N solution 40gr must be dissolved in 1ltr water or 1gr in 25ml water or 0.4gr in 10ml water.
Preferences:
[1] Prof. Dr. Peter Böck (1989, 17., neubearbeitete auflage), Romeis Mikroskopische Technik, ISBN: 3‐541‐11227‐1, München. Verleger Urban & Schwarzenberg.
[2] Ausgewählte Färbemethoden für Botanik, Parasitologie und Zoologie (Juli 2003), Waldeck GmbH & Co., 48161 Münster/Germany, Havixbeckerstrasse 62, see https://www.waldeck-ms.de/chroma-shop/ or in downloads
[3] Article number from the catalogue of the company Chroma in Münster (Germany), see https://www.waldeck-ms.de/chroma-shop/ or in downloads
[4] J.A. Schraag (1972), Handbook for teaching practical biology, uitgever: Muusses te Purmerend. ISBN: 9789063080396