(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Children's Library | Biodiversity Heritage Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

Full text of "Formulae and methods IV [i.e., 4th ed.] of the Marine Biological Laboratory Chemical Room"

MARINE 
BIOLOGICAL LABORATORY 



FORMULAE 

AND 

METHODS 

IV 



207 e J. 
M 29 



P 



WOODS HOLE, MASSACHUSETTS 






FORMULAE AND METHODS IV. 
OF THE 
MARINE BIOLOGICAL LABORATORY 
CHEMICAL ROOM 
Gail M. Cavanaugh, Editor 



a i 
Lj : 
a ' 
fc-" i 

a = 
a I 
jT ; 

U" i 

J ! 

UJ ! 



•3 

■ 09 

— ^ 
■o 




Copyright 1956 by the Marine Biological Laboratory, Woods Hole, Massachusetts. 
All rights reserved. This book, or parts thereof, may not be reproduced in any 
form without permission of the publishers. 



i 



PREFACE TO FOURTH EDITION 

Our Formulae and Methods Manual has been prepared from information 
acquired by our Chemical Room Staff over the course of more than 25 
years. The first, second, and third editions were prepared and edited 
by Oscar W. Richards. The first edition was published as a supplement 
to The Collecting Net on August 30, 1930. It contained 12 pages of 
information on biological solutions, stains, buffers and photographic 
solutions. The second edition, containing additional information on 
the above subjects, was published on August 27, 1932. The Formulae 
and Methods Manual was completely revised and published as the third 
edition by Oscar W. Richards in 1936. 

During the past 15 years more information regarding artificial 
sea water, buffers, and other biological solutions and formulas has been 
obtained by our staff. This information has resulted in publication of 
The Fourth Edition. This edition contains a complete revision of the 
sections on buffers, artificial sea water, and photographic solutions. 
Several new tables have been added. It is interesting to note that the 
material on biological stains has remained practically unchanged during 
the years. A table of contents has been added for quick references. 

The editor wishes to express his thanks to J. D. Ostrow for his 
work on Chapters 2, 3, 7, 8 and 9; to Mary Kapp for her work on stains; 
to J. B. Russell for his assistance on buffers; to A. Bickel for his 
work on primary and secondary standards; and to E. B. Harvey for her 
work on the artificial sea waters. 

G.M.C. , 1954 



CONTENTS 

CHAPTER PAGE 

I, General Information 1. 

II. General Formulae 7. 

III. Killing and Fixing Fluids 13. 

IV. Stains and Staining Solutions 17. 

V. Standard Chemical Solutions, Stock Solutions, 

and Solubilities 27. 

VI. Indicators and Indicator Solutions 37. 

VII. Buffers 39. 

VIII. Saline and Artificial Sea Water Solutions 51. 

IX. Photographic Solutions 57. 



CHAPTER I. 
GENERAL INFORMATION 

PURITY OF CHEMICALS: 

Several grades of many of the chemicals are kept in stock and care 
must be used in issuing chemicals so that the proper quality is furnished 
to the investigator. If there is any doubt as to the quality or quantity 
requested, consult with the person in charge before filling the order. 

Every precaution must be taken to prevent contamination of the U.S. P. 
and Reagent grades. The necks and caps of all reagent bottles should be 
free from dust before the bottle is opened. Spatulas are not to be intro- 
duced into the reagent stock bottles unless necessary, and then only after 
they have been thoroughly cleaned and dried. Material removed is not to , 
be returned to the stock bottles of the higher grade chemicals. When 
an amount is issued in other than the original container the label must 
contain the name of the chemical, the name of the manufacturer, the grade 
and the lot number. Metal spatulas are not to be used in handling mer- 
curic chloride, iodine, silver nitrate, and other corrosive chemicals. 
When in doubt use glass or porcelain spoons. The weighing papers are to 
be used only once and all tools immediately washed and placed where they 
will drain and dry. Any chemical that is spilled is to be cleaned up 
immediately and put into the proper receptacle. 

The commercial or technical grade is satisfactory for many purposes 
such as freezing mixtures and cleaning fluids. U.S. P. chemicals have 
been prepared to meet the standards of the United States Pharmacopoea (qv) 
and while suitable for medicinal use may contain other impurities harmless 
for this purpose. The so-called chemically pure (C.P.) grades are more 
or less pure but as there are no generally accepted standards for these 
grades, the purity will vary with different lots and samples from differ- 
ent manufacturers. This grade is useful when the highest purity is not 
required. 

The purest chemicals commercially obtainable are further purified 
and are accompanied with an analysis indicating the tolerances or limits 
of certain impurities contained. The standards are based on those estab- 
lished by the American Chemical Society or given in Murray, Standards 
and Tests for Reagent Chemicals , Van Nostrand. No information is avail- 
able for other impurities not tested for and when the investigator is 
in doubt it is essential that he make the necessary tests or further 
purify the chemicals in accordance with his requirements. Since the 



-1- 



analyses are only limits of tolerance they are of little use in comparing 
different brands of chemicals which may contain varying amounts of other 
impur it ies . 

The purest grades include Me r c k ' s " Reagent Grade", Mai inckrodt ' s 
" Analytical Reagent ", Eimer and Amend' s "Tested Purity", Baker's 
" Analyzed" , etc. 

Reagent grade and special chemicals are to be issued only when 
this grade of purity is requested on the order. 

The confusion of different grades of materials is to be guarded 
against. For example, do not issue immersion oil for clearing oil just 
because both are different kinds of cedar oil. Certain of these confusing 
substances are specially marked. In the case of expensive materials issue 
no more than the amount marked on the bottle unless permission of the 
person in charge is given for a greater amount. 

SOLUBILITIES: 

If a special solution calls for a large amount of a material consult 
the table of solubilities, Chapter 5, or for stains. Chapter 4, to see 
if solution is possible. The Merck Index, Chemical Rubber Handbook of 
Chemistry and Physics, Lange's Handbook of Chemistry, International 
Critical Tables , and the Dictionary of Chemical Solubilities are excel- 
lent reference books concerning solubilities of compounds. If in doubt, 
consult with the person in charge before attempting the preparation. 
This may avoid an error and the wastage of much material. Chloretone, 
in particular, cannot be made stronger than 0.6% in water at room 
temperature . 

ACCURACY AND DECIMALS: 

Note that accuracy is relative. An error of 0.1 gram in 500 grams 
is only a 0.02% error, while the same error in 1 gram is a 10% error. 
Use the proper scales or balance for the particular job to be done. If 
there is any question as to the accuracy required in an order, consult 
with the investigator placing the order or with the person in charge. 
When a decimal is to be placed on the label of any preparation conform 
to the following rule: Write the decimal to the number of places known 
to be correct, and only one doubtful figure. This same rule can be 
applied to figures where the known value does not extend to decimals 
by writing one doubtful figure and the rest zeros. 

ACCURACY AND ERRORS: 

Absolute errors x, -x are deviations from the correct values and 
their sign is important for correct statement. They are expressed as 



2- 



correct to two decimals, or to the nearest million, etc. Absolute errors 
are more important in addition and subtraction: e.g., in a column of 
figures the absolute errors in tlie third place of a sum or a difference 
may be great enough to make the second place unreliable. Relative errors 
(x, -x)/x are connected with the number of significant figures and are 
usually expressed as percentages. These errors are important in multi- 
plication and division. In a product or quotient the number of signif- 
icant figures is equal to the number in the weakest factor. Many solu- 
tions need not be prepared more carefully than 5% while others must be 
made with care to insure sufficient accuracy. If in doubt as to the 
precision required consult with the investigator or with the person in 
charge. This information and that given above is to be used as a guide 
by the staff in the use of the equipment in the Chemical Room. 

ALCOHOL DILUTION: 

For ordinary histological work special strengths of alcohol may 
be prepared by taking the number of milliliters of 95% alcohol equal to 
the strength desired in a graduate and adding enough distilled water to 
make 95 ml. (Example: to prepare 60% take 60 ml. of 95% alcohol and 
add 35 ml. of distilled water making 95 ml. of the strength of the 
alcohol used . ) 

CLEANING METHODS: 

Scrubbing with a 2% solution of alconox and hot water followed 
by a liberal rinsing with tap water will remove most chemicals. A 
small amount of trisodium phosphate applied with the fingertips or with 
a small brush will remove pencil markings, most greases, xylene, and 
films of paraffin. No abrasives should be used in cleaning volumetric 
glass ware. When cleaning solution (sulfuric acid-dichromate ) is used 
continued rinsing is necessary. Ten rinsings with water, seven of 
tap and three of distilled, are necessary for adequate removal of the 
dichromate from the surface of the glass. Since chromic acid is toxic 
to living organisms, this is important. For many purposes 1 part con- 
centrated nitric acid to 9 parts water is superior to chromic acid as 
the nitric acid oxidizes organic material without leaving an adsorbed 
residue on the glass. Aqua regia will remove what can be removed by 
cleaning solution and will wash off completely with tap water. To clean 
staining jars use a little dilute hydrochloric acid. For the few dyes 
not removed by this, use a strong solution of sodium hydroxide. Another 
useful cleaning fluid is 1-5% trisodium phosphate. Rubber stoppers 
may be cleaned by boiling in dilute sodium hydroxide, then rinsing with 



-3- 



water, followed by boiling in dilute hydrochloric acid and finally 
tlioroughly rinsing with water. 

GLYCINE or GLYCOCOLL is an amino acid used medicinally and is not to 
be confused with the poisonous photographic developer glycin, 
(p-hydroxyphenylaminoacetic acid). 

A MOLAL SOLUTION (m) contains one gram-molecular weight dissolved in 
1000 grams of solvent. For ordinary aqueous solutions 1 ml. of water 
is used as 1 gram. For other solutions calculate according to density 
at the temperature used. 

A MOLAR SOLUTION (M) contains one gram-molecular weight in one liter 
of solution. Dissolve the material in less than one liter and make up 
to one liter in a volumetric flask. 

A NORMAL SOLUTION (acidimetry or oxidimetry) contains one equivalent 
of the active reagent in grams in one liter of solution. The equivalent 
value of any reagent will depend upon the conditions under which the 
reagent is employed. It may or may not be the same as a molar solution. 

PERCENTAGE SOLUTIONS: Percent means parts in one hundred parts. These 
solutions may be made up according to weight, volume, or any combination 
of these. For example, a 3% solution of KCl contains 3 grams of the salt 
in 100 grams of solution, or in 97ml. of water. A 3% solution can be 
made up in any one of three ways: (a) 3 grams of KCl in a total volume 
of solution of 100 ml.; (b) 3 grams in 100 ml. of water; (c) 3 grains in 
97 grams of water. The third method will give precisely a 3% solution. 
The first two methods do not give a 3% solution, but for concentrations 
of 3% or less the error is too small to be of significance. For percent- 
ages greater than 3% it is best to prepare the solution on the basis of 
weight. For example, a 40% solution of NaOH is made by adding 40 grams 
of NaOH to 60 grams of water. Some substances, e.g., alcohol, vary in 
strength according to percent by weight or volume. Percentage solutions 
(by weight) may be prepared with the solution balance. Place the bottle, 
or bottle and funnel, on the pan and balance by means of the weight on 
the ungraduated beam. Set the weight on one of the graduated beams and 
weigh out the solute, then set for the amount of the solution and add 
the solvent until the scale is balanced. The beams are graduated to 
facilitate the preparation of percentage solutions but the balance 
may be used to advantage for the preparation of other solutions. 

The dilution of percentage solutions (aqueous solutions by weight) 
can be accomplished easily by taking the number of milliliters 



(or multiples thereof) of the stock solution equal to the strength 
solution desired and adding enough distilled water to make the total 
number of milliliters equal to the strength of the stock solution. 
Examples, (a) to prepare 7.1% from 18% stock solution use 7.1 ml. of 
the stock plus 10.9 ml. of water which makes a total of 18 ml. (b) to 
obtain a 0.02% solution from a 0.4% stock solution use 1 ml. of stock 
(50 X 0.02) and 19 ml. of water (50 x 0.38) making 20 ml. (50 x 0.02 + 
50 X 0.38) = (50 X 0.4) of the required solution. 

In general, remember that in diluting solutions the volume of the 
concentrated solution times its concentration is equal to the volume 
of the dilute solution times its concentration, or 

Vol. X Conc.= Volj,x Cone- 
To make a 3/8 M solution from a IM solution, take three parts of the 
IM solution and dilute to eight parts. For example, to make 500 ml. 
of a 3/8 M solution of NaCl from a IM solution take 3/8 of 500 or 
186.5 ml. of IM NaCl and dilute to 500 ml. To make 50 ml. of a 
1:10,000 solution requires 1:10,000 of 50 or 0.005 grams of the active 
ingredient made to 50 ml. with water. 

PROOF is the scale used for measuring the strength of alcohol. 
Absolute alcohol is 200 proof; and a mixture containing 50% alcohol 
by volume is 100 proof (U.S.A.) 

A proof-gallon contains an amount of alcohol equal to that in 
a gallon of proof spirit (100 proof). A gallon of proof spirit is 
one-half alcohol. Wine gallons multiplied by 1.9 equals proof gallons, 

DELIQUESCENT CHEMICALS. Bottle tops of the following chemicals 
should be dipped in paraffin. 



Acetamide 
Acid arsenic 
Acid chromic 
Ac id citric 
Acid monochloracet ic 
Acid s i 1 icotungst ic 
Acid trichloracetic 
Aluminum chloride 
Aluminum nitrate 
Ammonium acetate 
Ammonium fluoride 
Ammonium thiocyanate 
Barium bromide 
Barium chloride 
Beryllium chloride 
Beryllium nitrate 



Beryllium sulfate 
Calcium bromide 
Calcium chlorate 
Calcium chloride, 
Calcium chloride, 
Calcium nitrate 
Calcium oxide 
Cobalt sulfate 
Ferric chloride 
Ferric nitrate 
Ferrous chloride 
Iodides (most forms) 
Lithium bromide 
Lithium chloride 
Lithium salicylate 
Magnesium bromide 



Magnesium chloride 
Magnesium nitrate 
Manganese chloride 
anhyd Manganese sulfate 
cryst Mercuric nitrate 
Potassium acetate 
Potassium carbonate 
Potassium thiocyanate 
Sodium arsenate, cryst 
Sodium chlorate 
Sodium hypophosphite 
Sodium selenate 
Sodium sulfide 
Sodium sulfite, anhyd. 
Sodium thiocyanate 
Starch 
Zinc chloride 



-5- 



CHAPTER II. 
GENERAL FORMULAE 

NOTE: Use distilled water in all solutions. 

ADAMKIEWICZ REAGENT: (for proteins). Concentrated sulfuric acid 
1 volume, glacial acetic acid 2 volumes. Heat substance with this 
reagent. Reddish- violet color denotes proteins. 

AGAR: 2% may be used to solidify various solutions. 

ALCOHOL, ACID: 70% alcohol 99 ml., cone, hydroclilonc acid 1 ml. 

ALCOHOL, ALKALINE: 70% alcohol 99 ml., 0.02 grams sodium bicarbonate, 

AMANN'S LACTOPHENOL: Phenol 20 grams, lactic acid (1.21 sp.gr.) 
16.5 ml., glycerine 32 ml., water 20 ml. For herbarium specimens, 
soften first in 1:10 lactophenol and then to pure lactophenol. For 
algae use, lactophenol 5 ml., water 95 ml., cupric chloride and cupric 
acetate each 0.2 grams. For mounting fungi add to the lactophenol 
0.05 grams cotton blue. 

AMMONIACL\L SILVER SOLUTION: Dissolve 13 g. of silver nitrate in 
about 250 ml. of water, add enough cone, ammonium hydroxide to re- 
dissolve the precipitate whicli forms upon the first addition of the 
NH^OH and make the volume up to 500 ml. with water. 

ANILINE WATER: Shake 4 ml, of aniline in 90 ml. of distilled water 
for at least 15 minutes. Filter through a wet filter. Enough alcohol 
may De added to make it 20% alcohol, if a weakly alcoholic solution is 
des ired . 

BARFOED'S SOLUTION: (test for dextrose in presence of maltose) 
13.3 g. cupric acetate in 200 ml. water, and 5 ml. of 38% acetic acid. 

BELAR'S SOLUTION: Water 100 ml., sodium chloride 0.9 g., potassium 
chloride 0.02 g., calcium chloride 0.02 g. , sodium bicarbonate 0.02 g. 
To this 0.25 to 0.5 g. glucose may be added. 

BENECHE'S NUTRIENT SOLUTION: ( Algae-bluegreen ) Water 1 liter, ammo- 
nium nitrate 0.2 g., calcium chloride 0.1 g., potassium phospliate 
(dibasic) 0.1 g., magnesium sulfate 0.1 g., 1% ferric chloride 1 drop. 
For use dilute 2-9 times as required. (Courtesy Dr. Navez) 



BENEDICT'S SOLUTION - QUALITATIVE: Cupric sulfate 17.3 g. , Sodium 
citrate 173 g. , sodium carbonate 100 g. , water 1000 ml. Dissolve the 
sodium citrate and carbonate in 800 ml. water (filter if necessary). 
Dissolve the cupric sulfate in 100 ml. Add the cupric sulfate slowly to 
the citrate -carbonate solution with constant stirring and dilute to 1 
1 iter . 

BENEDICT'S QUANTITATIVE SUGAR REAGENT: Cupric sulfate 18 g. , sodium 
carbonate (1/2 weight of anhydrous salt may be used) 200 g., sodium or 
potassium citrate 200 g., potassium thiocyanate 125 g. , potassium ferro- 
cyanide (5% solution) 5 ml., water to make up volume to 1000 ml. With 
the aid of heat dissolve the carbonate, citrate, and thiocyanate in 
enough water to make 800 ml., and filter if necessary. Dissolve the 
cupric sulfate separately in 100 ml. and mix slowly with the other 
solution. 25 ml. of the reagent are reduced by 50 mg. of glucose. 

BRODIE'S SOLUTION: Water 500 ml., sodium chloride 23 g., sodium 
chlorate 5 g. , 1% (aq.) methylene blue 3 ml., thymol O.lg. 

CARBO-XYLOL: 1 part liquified phenol (see below), and 3 parts of 
xylene. 

CARLSBAD SALTS, SYNTHETIC: (Sprudelsalz ) Sodium sulfate 11 g., 
sodium bicarbonate 10 g. , sodium cliloride 9 g. , potassium nitrate 19 g. , 
potassium sulfate 1 g. 

CEMENT: Beeswax 58%, rosin 29%, Venetian turpentine 13%. 

CHALKLEY'S MEDIUM: Water 1000 ml., sodium chloride 0.1 g., potassium 
chloride 0.0004 g., calcium chloride 0.006 g. 

CHLORETONE: Chlorbutanol 0.6 g. soluble in 100 ml. water 20°C. 

CLARK'S FLUID: (for insect tissue culture) Water 200 ml., sodium 
chloride 1.3 g. , potassium chloride 0.028 g., calcium chloride 0.024 g. , 
sodium bicarbonate 0.02 g., monobasic sodium phosphate 0.002 g. 

CLEANING SOLUTION: Dissolve 60 - 65 g. , of sodium or potassium 
bichromate by heating in 30 - 35 ml. of water. Cool and slowly add con- 
centrated sulfuric acid to make a liter of solution. 

CLERICI'S SOLUTION: 10 ml. water, 50 g. thallium (ous) malonate, 50 g, 
thallium (ous) formate, 0.1% sodium t auroglychocholate . Keep cool and 
filter. Density about 4. 



-8- 



CZAPEK' S MEDIUM: (for molds) Sucrose 30 g., sodium nitrate 2 g., 
dibasic potassium phosphate 1 g., magnesium sulfate 0.5 g., potassium 
chloride 0.5 g. , ferrous sulfate 0.01 g. , water 1000 ml. 

FEHLING'S SOLUTION: Solution I. Cupric sulfate 34.65 g. in 500 ml. water. 

Solution II. Potassium hydroxide 125 g., Rochelle salts (sodium potassium 

tartrate) 173 g. , water to make 500 ml. Solutions mixed in equal volumes 
for use. 

FIESER'S FLUID: Water 100 ml., sodium hydrosulfite 16 g., sodium 
hydroxide 13.3 g., sodium anthraquinone-B-sulf onate 4 g. 

FILM PRESERVATIVE: (acetate base) Camphor 4 g., menthol 4 g., oil 
eucalyptus 8 g., glycerine to make 125 ml. 

GLYCERINE JELLY: Water 60 ml., gelatin 10 g. , glycerine 70 ml., phenol 
(cryst.) 0.25 g. Soak gelatin 30 minutes in the water, dissolve with 
gentle heat. Add glycerine and phenol and stir until homogeneous. Store 
in wide mouth bottle. 

GRAMS IODINE: Water 300 ml., potassium iodide 2 g. , iodine 1 g. ' Mix 
KI and I2 in a few drops of water. When dissolved add the remaining 
quantity of water. 

GREEN FILTER SOLUTION: Water 300 ml., cupric sulfate 35 g. , potassium 
dichromate 3.5 g. , cone- sulfuric acid 1 ml. 

GROUND GLASS SUBSTITUTE: Ether 240 ml., sandarach 12 g. , mastic 2.6 g., 
benzene 160 ml. 

HAINES SOLUTION: Water 170 ml., cupric sulfate 2 g. , potassium hydrox- 
ide 6.7 g. , glycerine 15 ml. 

HAYEM'S SOLUTION: (for microscopic examination of blood) Water 1000 
ml., mercuric chloride 2.5 g. , sodium sulfate (anhyd) 25 g. , sodium 
chloride 5 g. 

HEAT ABSORBING FLUID: 2% aqueous calcium chloride. 

HEAT ABSORBING FLUID: Water 1000 ml., Mohr's salt (ferrous ammonium 
sulfate) 200 g. Dissolve and filter. If not clear, add 1.7 ml. cone, 
sulfuric acid. 

HOLTFRETER'S SOLUTION: Water 1000 ml., sodium chloride 3.50 g. , pot- 
assium chloride 0.05 g., calcium chloride (anhyd) 0.10 g., sodium bicar- 
bonate 0.20 g. When this is to be sterilized do not add the bicarbonate 
until ready to use. 



-9- 



IODINE, TINCTURE: U.S. P. 70% Alcohol 1000 ml., iodine 70 g. , potassium 
iodide 50 g. 

IODINE, WATER: Water 1000 ml., iodine 0.2 g. 

KEEFE'S SOLUTION: (for preserving green organisms) Alcohol 50% 
90 ml., formaldehyde 5 ml., acetic acid (glacial) 2.5 ml., glycerine 
2.5 ml., cupric chloride 10 g. , uranium nitrate 1.5 g. 

KLOTZ SOLUTION: (for preservation of invertebrate animals) Chloral 
hydrate 50 g., Carlsbad salts (see above) 50g. , formalin 100 ml., water 
to make 1000 ml. 

KNOP'S SOLUTION: Water 1000 ml., calcium nitrate 1 g., potassium 
chloride 0.25 g., magnesium sulfate 0.25 g. , monobasic potassium phos- 
phate 0.25 g., ferric chloride trace. 

KNOP'S SOLUTION: (modification) Water 1000 ml., potassium nitrate 
1 g. , calcium sulfate 0.5 g. , magnesium sulfate 0.5 g. , calcium phos- 
phate 0.25 g. , ferrous phosphate 0.25 g. 

LOCKE'S SOLUTION: (for warm blooded animals ) Water 1000 ml., sod- 
ium chloride 9 g., calcium chloride (anhyd) 0.24 g. , potassium chloride 
0.42 g. , sodium bicarbonate 0.2 g. , dextrin 2.5 g. 

LUGKDL'S IODINE: Water 100 ml., iodine 4 g. , potassium iodide 6g'. 
Mix KI and I2 with a few drops of the water. When dissolved, add the 
remaining quantity of water. 

MAYER'S ALBUMEN FIXATIVE: Separate the white albumen and cut it 
with scissors to break up the large masses. Filter through coarse 
filter paper and add an equal volume of glycerine. A crystal of thymol 
acts as a preservative. 

MASSART'S MOUNTING MEDIUM: Water 100 ml., glycerine 16 ml., chloral 
hydrate 100 g. , gum arabic 50 g. After fixing in distilled water for 
24 hours, then 24 hours in 50% chloral hydrate followed by 24 hours in 
100% chloral hydrate and then mounted in the above solution. (Courtesy 
Dr. Navez) 

MILLON'S REAGENT: (for proteins and nitrogenous compounds) Mercury 
10 g., concentrated nitric acid 15 ml. Dissolve the mercury in the 
acid, then dilute the solution with 2 volumes of water. Let stand 24 
hours and decant. Gives a red color with proteins. 

MIQUEL'S SOLUTION: I (culture fluid for diatoms) A. Water 100 ml. 
magnesium sulfate 10 g., sodium cliloride 10 g., sodium sulfate 5 g., 

-10- 



ammonium nitrate 1 g., potassium nitrate 2 g., sodium nitrate 2 g., 
potassium bromide 0.2 g., potassium iodide 0.1 g. B. * Water 80 ml., 
sodium phosphate 4 g., calcium chloride (anhyd) 4 g., cone, hydrochloric 
acid 2 ml., ferric chloride 2 g. 

•Make as follows: To the sodium phosphate dissolved in 40 ml. of 
water are added, first 2 ml. of HCl , then 2 grams of ferric chloride and 
then 4 grams of calcium chloride dissolved in 40 ml. of water, taking 
care to shake the mixture. There will be a brown precipitate of ferric 
oxide on adding the last solution. It should be separated from the 
liquid before using. Forty drops of solution A and 10 - 20 drops of 
solution B are added to each 1000 ml. of sea water sterilized by keeping 
at 70 C for about 20 minutes. 

MIQUEL'S SOLUTION: II (May be added to A and B) Sodium silicate 
•9 HgO 5 g. , water 100 ml. 

MIQUEL'S SOLUTION: III (Allen's modification) A. Water 100 ml., po- 
tassium nitrate 20.2 g. B. Water 80 ml., dibasic sodium phosphate 
4 g. , CaCl2»6 H2O 4 g. , ferric chloride (melted) 2 ml., cone. HCl 2 ml. 
For use, add 2 ml. of solution A and 1 ml. of solution B to each 1000 
ml. of sea water. 

MOORE'S SOLUTION: Water 1000 ml., ammonium nitrate 0.5 g. , monobasic 
potassium phosphate 0.2 g. , magnesium sulfate 0.2 g. , calcium chloride 
(anhyd) 0.1 g. , ferric sulfate trace. 

NAEGELI'S SOLUTION: (culture medium for fungi) Water 1000 ml., di- 
basic potassium phosphate 1 g. , magnesium sulfate 0.2 g., calcium 
chloride (anhyd) 0.1 g., ammonium tartrate 10 g. 

NESSLER'S REAGENT: (Block and Benedict from Hawk and Bergein) 
Mercuric iodide 100 g., potassium iodide 70 g. , sodium hydroxide 100 g. 
Place 100 g. mercuric iodide and 70 g. of potassium iodide in a liter 
volumetric flask and add about 400 ml. of water. Rotate until solution 
is complete. Now dissolve 100 g. of sodium hydroxide in about 500 ml. 
of water, cool thoroughly and add with constant shaking to the mixture 
in the flask, and make up with water to the liter mark. If a precip- 
itate forms, decant the supernatant liquid and use. 

PASTEUR'S SOLUTION: Potassium phosphate 2 g., calcium phosphate 0.2 g., 
magnesium sulfate 0.2 g., ammonium tartrate 10 g., cane sugar (sucrose) 
150 g., water to make 1 liter. 

PHENOL: To liquify add 10 ml. of water to each 450 g. melted phenol. 
Melt by immersing bottle in hot water. 

-11- 



PLATING SOLUTION FOR ELECTRODES: Platinum chloride 1 - 3%, lead ace- 
tate 0.02%. Use 4 volts for about 10 minutes. 

PYROGALLOL FLUID FOR ABSORBING OXYGEN: Water 500 ml., potassium 
hydroxide 220 g., pyrogallic acid 15 g. 

POISON IVY PREVENTIVES: I. Saturated aqueous sodium thiosulfate. 

II. Ferric chloride 5 g. , glycerine 50 ml., water 50 ml. 

III. Ferric chloride 5 g., glycerine 5 ml., 50% alcohol 100 ml. 

RAFFEL'S FLUID: Water 1000 ml., potassium nitrate 0.5 g. , dibasic 
potassium phosphate 0.06 g., magnesium sulfate 0.02 g., ferric chlo- 
ride 0.001 g. 

RINGERS SOLUTIONS: Cf. table in Chapter VIII 

SALINE: (normal physiological) Water 1000 ml., sodium chloride 
7 to 9 grams. For cold blooded animals, use 7 g. For warm blooded 
animals use 9 g. 

SZOMBATHY'S FLUID: Gelatin 1 g. , water 100 ml., sodium silicate 
0.02 g. , glycerine 15 ml. Dissolve the gelatin at 30 C. Cool and 
filter through cloth. 

TOISSON'S MIXTURE: (a diluting fluid for blood) Water 160 ml., 
sodium sulfate 8 g., sodium chloride 1 g., glycerine 30 ml, j methyl 
violet 0.025 g. 

TYRODE'S SOLUTION: (for gut muscle, no advantage for heart muscle) 
Water 1000 ml., sodium chloride 8 g. , potassium chloride 0.2 g., 
calcium chloride (anhyd) 0.2 g., magnesium chloride 0.1 g., monobasic 
sodium phosphate 0.05 g., sodium bicarbonate 1 g. , glucose 1 g. 

ULTRA-VIOLET LIGHT is removed by saturated aqueous sodium nitrate 
or 10% CuS04»5 H2O. 

VAN DER CRONE'S SOLUTION: (green organisms) Water 1 liter, pot- 
assium nitrate 1.0 g., magnesium sulfate and calcium sulfate each 
0.5 g., calcium phosphate 0.25 g. , ferrous phosphate 0.25 g. Make 
slightly acid or neutral with phosphoric acid. 

VAN'T HOFF'S SOLUTION: (artificial sea water) Sodium chloride 19.0 
g. , magnesium sulfate 1.5 g., magnesium chloride 2.4 g., potassium 
chloride 0.53 g. , calcium chloride (anhyd) 0.37 g., anhydrous salts 
dissolved and made up to 1000 ml. with glass distilled water. Also 
Cf. table in Chapter VIII. 



-12- 



CHAPTER III. 

KILLING AND FIXING FLUIDS 

COPPER ACETATE FORMALIN: Saturated cupric acetate in 40% formalde- 
hyde. Dilute to about 4% for preservation of green algae. 

CUPRIC-PARANITROPHENOL FLUID: (Petrunkevitch) 60% alcohol 100 ml . , 
nitric acid 3 ml., ether 5 ml., cupric nitrate (3 HpO) 2 g. , para- 
nitrophenol cryst. 5 g. One part formaldehyde may be added to 4-7 
parts of the fluid just before use. 

CUPRIC PHENOL FLUID: (Petrunkevitch) _A^: distilled water 100 ml., 
nitric acid 12 ml., cupric nitrate (3 HgO) 8 g. _Bj 80% alcohol 100 
ml., phenol cryst. 4 g. , ether 6 ml. Use 1 part of j\^ to 3 of B. 
Will not keep after mixing. 

FAA: (General Biological Supply House) 50% alcohol 100 ml., 40% 
formaldehyde 6.5 ml., glacial acetic acid 2.5 ml. 

HOLLANDE'S FLUID: Formalin sat. with picric acid 12 ml., absolute 
alcohol 54 ml., benzene 3 ml., and nitric acid 1 ml. 

LANE'S SOLUTION: Potassium bichromate 2.5 g., mercuric chloride 
5.0 g. , water 100 ml. 

NAVASCHIN'S FLUID: 10% chromic acid, 1.5 ml., glacial acetic acid 
1 ml., formaldehyde (40%) 0.83 ml., water 32.67 ml. 

PRESERVING FLUID FOR GREEN ALGAE: Potassium chrom alum 10 g.j form- 
aldehyde 5 ml., water 500 ml. 

STOCKARD'S SOLUTION: Water 85 ml., formalin 5 ml., glacial acetic 
acid 4 ml., glycerine 6 ml. 

SUSA'S FLUID: Water 80 ml., mercuric chloride 4.5 g., sodium chloride 
0.5 g., trichloracetic acid 2.0 g., formalin 20 ml., glacial acetic 
acid 4 ml. 

WORCESTER'S FLUID: 10% formalin saturated with mercuric chloride, 
90 ml., glacial acetic acid, 10 ml. 

OSMIUM AND PLATINUM CONTAINING FLUIDS: 

Osmium and platinum fixatives are costly and often do not keep 



13- 



well. Few cytologists use the same formulae, each usually wanting 
his favorite formula, hence it is best to keep on hand certain stock 
solutions, among which are small amounts of osmic acid and platinic 
chloride. Below are listed certain fixatives containing one or both 
of these reagents, also a list of stock solutions. The makeup of the 
fixatives from the stock solutions is given in parts by volume, and the 
amount desired by an investigator can be made up to the nearest mul- 
tiple of the total parts indicated. 



Acetic acid, glacial 

Chromic acid, 1% 

Chromic acid, 1% in 

1% NaCl 

Formic acid 

Mercuric chloride, sat 

soln. in hot water 



STOCK SOLUTIONS 

Mercuric chloride, 0.5% 
in 1% chromic acid 
Osmic acid, 2% 
Picric acid, sat. aq.soln. 
Platinic chloride, 10% 
Potassium dichromate, 10% 



FIXATIVES: 

Some of the fixatives listed here keep well and may be kept for 
a long time. Those which deteriorate are noted. All of these form- 
ulae are from Lee's Vade Mecum, unless otherwise stated. In making 
osmic acid wash off the paper covering of the glass ampoule; rinse 
in distilled water, and file a notch around the tube. Drop the am- 
poule into a clean, glass stoppered bottle of a capacity greater 
than the amount of osmic desired. The tube of osmic crystals may 
now be broken open with a heavy glass rod. As many tubes as wanted 
may be crushed inside the glass bottle but not over 200-300 c.c. of 
2% should be kept in solution. 

All osmic acid and fixatives containing it should be kept in 
dark bottles with well fitted glass stoppers. Osmic acid reduces 
slowly in the light and at high temperatures; when it is issued it 
should be in a brown bottle or the bottle should be covered with black 
paper . 

When issuing fixatives the label should indicate definitely the 
formula used, since there are 4 Flemming's and 3 Von Rath's solutions. 



14- 



— ' CM 



t) 

B 

s 





c^ 


«^ 








3 


a 




60 


M 






ss: 


1 


4) 


g 


-* 


a 

E 


>> 


►— « 




o 


0} 




s 


u. 


-o 


D 


s? 


o 




fS 


CM 


■H 








to 


<J 


^ 


s 


O 


< 


HH 


s? 








o 


s 




Q 


^H 


o 


-a 






(h 


•H 


g 


s 


6 


^ 


C/3 








hH 




u 





C^ 



C3 



Q. 13 



tis Q 



4^ o 

feS; — , 

o o 

o ^ 

—I o 



s < 



o < 



O « 



s < 



■T3 O 

c m 

CO (/} 

(J-T3 

O 

o— < 



o(r. 



T3T3 u 

10 >■ 
•— « JJ 

TT O O . 

C » C QJ 

(Q en v) 

.-i-O 3 
CJ-C — 

O 3 O 

O— ' O J-> 

4J C T3 

3 3 -D m 

O -H 4J 

-^ Lh u u 



01/) O-H 
• E C 

S n o 3 
u V u 
a usi-a 

* 3CJ V 



TS 




V 


•M 




-u 


o 






^ 




4J 




0) 


M 


o 


-o 


3 


• H 


■H 


■'-) 


(h 


tH 




Jj 


o 


OJ 


• H 




-o 


z 


6 


>- 


o 


E 


-o 


c 


3 




a 




CO 

E 




C^ 


u 

. 


^ 




<« 60 


:s 


60 


-O-H 


in 




T) m 




— ^ 


< 3 





o 


o 






•H 


sT 


6 


4-> 


CM 


E 


z 


X 


3 
•H 






-T3 


u 


o 





c 


c 


CAj 


o 


o 




a 


CJ 


• 





-a 

< 



3 



60 

C 



CM <— I 



CO 








u ^ 






4J (0 






c 




T) 


u C 




C 


o 


D 


-1 c 


■J CO 



I I 



o 
o 






o 

o 



c3 



Ltf m 



CQ 



03 



o 
c 
t-i 

6 



>- c 

O 3 

c u 

CO 0} 



60 



60 60 

c c 

•H O 



e E .0) E <-> 
CO v^ 0) c/J 

(5 S^ E""' 



a 



01 

B 



t2 






^ci? 



-:^ 



a 

o o — ■ 

h C3S I 
CO 



o 
o 



■HT3 
11 

CB 01 

Ht3 









•H <J 

Q.-H 






— ; CO 3 



K 

B 
O 

> 



13 

3 



S 



cJ5 f5 



-15- 



CHAPTER IV. 
STAINS AND STAINING SOLUTIONS 

NOTE: Use distilled water in all solutions. 

ACETO- CARMINE: (Schneider's) Mix equal volumes of glacial acetic 
acid and water. Saturate with powdered carmine, boil, cool and filter. 

ALUM COCHINEAL: Boil powdered cochineal in 5% aqueous solution of 
aluminum potassium sulfate or aluminum ammonium sulfate. Filter and 
add a little salicylic acid to the filtrate as a preservative. 

AMMONIA- CARMINE: (Ranvier) Dissolve carmine in water with a slight 
excess of ammonia. Evaporate to dryness, dissolve the residue in water, 
and filter. 

AZO-CARMINE: Azo-carmine GX 0.2 g. , boiling water 100 ml. When cool 
filter and add 1 ml. glacial acetic acid. The precipitate redissolves 
as the stain is used at 55 C. Azo-carmine B 0.33 g., water 100 ml., 
acetic acid trace. 

AZAN STAIN: cf. Mallory A' and Azo-carmine. 

BEST'S CARMINE: Carmine 2 g. , potassium carbonate 1 g. , potassium 
chloride 5 g., water 60 ml. Boil gently for a few minutes and cool. 
Then add 20 ml. of cone, ammonium hydroxide. 

BORAX -CARMINE: (Grenacher' s ) Sodium tetraborate (borax) 4% aqueous 
solution 100 ml., carmine 3 g. Boil until the carmine dissolves, cool 
and add 100 ml. of 70% alcohol. Filter after 24 hours. 

BORREL: A. 1% aqueous magenta (basic fuchsin)JB_. Indigo carmine 1 g., 
distilled water 60 ml., saturated aqueous picric acid 40 ml. 

CARBOL-FUCHSIN: Fuchsin (sat. ale. sol.) 10 ml., phenol (5% aq. sol.) 
90 ml. 

CARBOL-FUCHSIN: (Goodpasture's) Alcohol (20%) 100ml., phenol (melted) 
1 ml., aniline 1 ml., basic fuchsin 0.5 g. 

CARMALUM: (Mayer's) Carminic acid 1 g., aluminum ammonium or aluminum 
potassium sulfate 10 g., water 100 ml. Dissolve with heat and filter 
the solution when cold. Add a crystal of thymol as a preservative. 

ERLICH'S TRIPLE STAIN: (triacid mixture) Orange G (sat. aq. sol.) 



17- 



14 ml., acid fuchsin (sat. aq. sol.) 7 ml., water 15 ml., absolute 
alcohol 25 ml., methyl green (sat. aq. sol.) 12 ml., glycerin 10 ml. 
Each solution should be thoroughly saturated (several days). Add the 
ingredients in order named, shaking mixture well after each addition. 

FEULGEN'S REAGENTS: eg. nucleal reaction. 

GRAM'S STAIN: See Gential violet and Gram's iodine Chapter II. 

HEMALUM: (Mann's) liaematein 0.5 to 2 grams, absolute alcohol 100 ml., 
glycerine 100 ml., water 100 ml., potassium aluminum sulfate (potassium 
alum) 10 g. , glacial acetic acid 10 ml. Dissolve the dye in the acid, 
add the alcohol, glycerine and 75 ml. of the water; then dissolve the 
alum in the rest of the water with gentle heat and add to the other 
ingredients. Five grams of aluminum sulfate may be substituted for the 
potassium alum. 

HEMALUM: (Mayer's) Hematoxylin 1 g. , water 1 liter. Dissolve and 
add sodium iodate 0.2 g. , ammonium aluminum sulfate or potassium alumi- 
num sulfate 50 g. Heat to dissolve. When cool, filter. 

HEMALUM, ACID: (Mayer's) To Mayer's hemalum add glacial acetic acid 
to 2%. 

HEMATOXYLIN STOCK SOLUTION: May be made by dissolving 1 part of hem- 
atoxylin crystals in 10 parts of absolute alcohol. In the course of 
several months or a year, this solution ripens to a dark wine-red color. 
It may be used in making up the various hematoxylin solutions, and being 
ripe, will stain at once. 

HEMATOXYLIN: (Delaf ield' s ) Aluminum ammonium sulfate (ammonium alum) 
saturated aqueous solution 100 ml. Dissolve 1 g. of hematoxylin crystals 
in 10 ml. of absolute alcohol, and slowly add it to the ammonium alum. 
Expose to air and light for several weeks to 'ripen'. Ripening may be 
accomplished at once with some degree of success through the addition of 
a few ml. of hydrogen peroxide. When ripe, filter the solution and add 
25 ml. of glycerine and 25 ml. of methyl alcohol. (Cf. Hematoxylin stock 
solution ) . 

HEMATOXYLIN: (Erlich's acid) Hematoxylin 2 g., absolute alcohol 100 ml., 
glacial acetic acid 10 ml., glycerine 100 ml., water 100 ml., aluminum 
ammonium sulfate or aluminum potassium sulfate 10 g. Let the mixture 
ripen in the light and air until it acquires a dark red color. 



-18- 



m 



HEMATOXYLIN: (Galigher's) Hematoxylin crystals 0.5 g., aluminum 
ammonium sulfate 0.3 g., 50% alcohol 100 ml., red mercuric oxide 0.6 g. 
Dissolve hematoxylin and alum in the alcohol. When boiling add mercuric 
oxide and boil for 20 minutes in a covered vessel. Let stand overnight. 
Filter. Solution can be used at once. 

HEMATOXYLIN: (Harris's) Dissolve 20 g. of ammonium or potassium alu- 
minum sulfate in 200 ml. of water with the aid of heat. Dissolve 1 g. 
of hematoxylin crystals in 10 ml. of absolute alcohol and add to the war 
alum solution. Bring rapidly to boil and add 0.5 g. of red mercuric oxide 
When the solution turns dark purple, cool, and add 8 ml. of glacial acetic 
acid. 

HEMATOXYLIN: (Heidenhain' s ) (iron-hematoxylin) _A. Ammonium ferric 
sulfate (ferric alum) 2.5 g., water 100 ml. _B. 10% Hematoxylin stock 
solution 5 ml., distilled water 100 ml. 

HEMATOXYLIN: (Mann's) M&ke up Erlich's Hematoxylin stain with haem- 
atein instead of hematoxylin. 

HEMATOXYLIN: (Mayer's) Hematoxylin stock solution 10 ml., sodium 
iodate 0.26 g., chloral hydrate 6.6 g., aluminum ammonium or aluminum 
potassium sulfate 1.2 g. , water to 2 liters. 

HEMATOXYLIN: (Regaud's) Hematoxylin stock solution 10 ml., glycerine 
10 ml. , water 80 ml. 

INDIGO- CARMINE: Indigo-carmine 0.2 g., water 100 ml. 

LOEFFLER'S METHYLENE BLUE: Methylene blue 0.3 g. , 95% ethyl alcohol 
30 ml., 0.01% potassium hydroxide 100 ml. 

MALLORY'S TRIPLE STAIN: (for connective tissue) _\^ Acid fuchsin 0.5 g, 
water 100 ml. _B. Anilin blue (Gruebler's water soluble) 0.5 g. , Orange G 
(Gruebler) 2 g. , 1% aqueous phosphomolybdic acid 100 ml. 

MALLORY'S SOLUTION A': (also called Heidenhain' s azan stain) 0.5% 
Azo-carmine with 5-10 drops of glacial acetic acid to each 100 ml. of 
solution. Do not filter. Cf. also Azo-carmine. (Courtesy of Mrs. N. 
Jones ) 

MUCI-CARMINE: (Mayer) Water 2 ml., carmine 1 g. , aluminum chloride 
0.5 g., 50% alcohol 100 ml. Mix in order given, heat gently until the 
fluid darkens (about 2 minutes); filter after 24 hours. To use, dilute 
with 5 to 10 volumes of water. 



-19- 



NUCLEAL REACTION: Feulgen's modification of the Schiff reaction for 
aldehydes. _A. Water with an excess of sulfur dioxide. Water 200 ml., 
sodium bisulfite 1.3 g., IN hydrochloric acid 10 ml. B. Fuchsin- 
sulfurous acid reagent . Dissolve 1 g. of basic fuchsin in 100 ml. warm 
water. Filter. Add 20 ml. of 1 N hydrochloric acid and 1 g. sodium 
bisulfite. Let stand 24 hours. Decolorize with Norite and filter. 

PARACARMINE: (Mayer's) Carminic acid 1 g., aluminum chloride 0.5 g. , 
calcium chloride 4 g., 70% alcohol 100 ml. Dissolve and allow to settle. 
Filter, 

PICRO-CARMINE: Cone, ammonium hydroxide 5 ml., water 50 ml., carmine 
1 g. When dissolved add saturated aqueous picric acid SO ml. Expose 
to air and light for 2 days, then filter. 

PICRO- FUCHSIN: (Van Gieson) Acid fuchsin (1% aq. sol.) 10 ml., 
picric acid (sat. aq. sol.) 90 ml. 

POLYCHROME METHYLENE BLUE: (for staining cell granules) 
Michaelis ' method : Methylene blue 2 g. , water 200 ml. To this solution 
add 10 ml. of 0.1 N sodium hydroxide. Boil for 15 minutes. After cool- 
ing add 10 ml. of 0.1 N sulfuric acid and filter. 

POLYCHROME METHYLENE BLUE: Unna's method : Methylene blue 1 g. , potas- 
sium carbonate 1 g., 95% alcohol 20 ml., water 100 ml. Evaporate to 
100 ml. It may be used at once, or after diluting with an equal volume 
of anilin water. See Chapter III. 

SCHARLACH R: 70% alcohol 50 ml., acetone 50 ml., Scharlach red to 
saturation. 

THIONIN, ACID: (Frost's form) Thionin 1 g. , phrenol 2.5 g. , glacial 
acetic acid 20 ml., water 400 ml. 

TRIACID STAIN: See Erlich's triple stain. 

UNNA-PAPPENHEIM STAIN: (modified) Pyronin Y 0.9 g. , methyl green 
0.1 g., 95% alcohol 9 ml., glycerine 10 ml., 0.5% phenol to 100 ml. 

WEIGERT'S STAIN: (for elastic tissue) (resorcin-fuchsin) Basic 
fuchsin 2 g. , resorcinol 4 g. , water 200 ml. Heat the mixture in a 
porcelain dish and while boiling add 25 ml. of a 29% aqueous solution 
of ferric chloride (FeCl3»H20). Stir and boil for 2-5 minutes. A pre- 
cipitate forms. Filter. Discard the filtrate. Drain the filter paper 
dry and return the paper and precipitate to the dish. Add 200 ml. of 



-20- 



95% alcohol and boil, stirring constantly. Remove the paper from the 
solution, filter, and add alcohol to make the solution up to 200 ml. 
Add 4 ml. of cone, hydrochloric acid. The solution keeps well for 
months . 

WRIGHT'S STAIN: Wright's stain (dry powder) 0.2 g., methyl alcohol 
(absolute, neutral, acetone free) 60 ml. Filter after standing for 
24 hours. 

STAIN SOLUBILITIES 

References : Conn, Biological Stains, 1946; Holmes, Stain Technology, 

1929 

Dye solubilities at 26*'C listed as grams of anhydrous dye per 100 ml. 

of saturated solution selected from the above references and printed 

with the permission of Dr. H. J. Conn. 

Aniline dyes are commonly used as saturated solutions, aqueous, or 
alcoholic, unless other concentrations are given. 

KEY TO BIOLOGICAL USE OF THE STAINS: 

B - bulk N - nuclear 

C - cytoplasmic P - perfusion 

F - fat V - vital 

I - indicator 

Synonyms of the dyes are given in parentheses. 

Color, 95% Strength Use 

Index Name of Dye Water ,, , , , . 

Alcohol solution 



Number 



used 



1027 Alizarin Nil 0.125 I.C.V. 

1034 Alizarin red S (Alizarin red, water 

soluble; Alizarin carmine) 7.69 0.15 N.V. 

40 Alizarol orange G (Alizarin yellow 
R; Mordant yellow PN; Orange R; 
Anthracene yellow RN; Alizarin 
orange) 0.40 0.57 

36 Alizarin yellow GW (Alizarin yellow 
GG; Anthracene yellow GG; Mordant 
yellow 2GT) 25.84 0.04 

184 Amaranth (Naphthol red S,C or 0; 
Fast red; Bordeaux; Bordeaux 
SF; Victoria rubin 0; Azo rubin; 

Wool red) 7.20 0.01 C. 

847 Amethyst violet (Heliotrope B; 

Iris violet) 3.12 3.66 N. 



■21- 



Color Strength 

Index Name of Dye Water 95 7c Solution Use 

Number Alcohol Used 



655 Auramin (Canary yellow; Pyok- 

taninum aureum; Pyoktanin 

yellow) 0.74 4.49 V.N. 

12 Aurantia (Imperial yellow) Nil 0,33 C. 

724 Aurin (Rosolic Acid) 0.12 40.0 I. 

146 Azo acid yellow 2.17 0.81 

448 Benzopurpurin 4B (Cotton red 48; 

Dianil red 43; Diamin red 48; 

Sultan 48; Direct red 48) 0.13 C,V. 

280 Biebrich scarlet (Croceine scarlet 

5R; Ponceau 8; Double scarletBSF; 

Scarlet 8, or EC) 0.05 C. 

332 Bismarck brown R (Bismarck brown 

GOOO; Brown R.AT.C or N; Man- 
chester brown EE; Vesuvin NR,B, 

R; Basic brown BR or BXN) 1.10 0.98 N. 

331 Bismarck brown Y (Vesuvin; Phenyl- 

ene brown; Manchester brown; 

Excelsior brown; Basic brown G, 

GX, or GXP) 1.36 1.08 V.C.B. 

88 Bordeaux red (Fast red B, BN or P; 

Cerasin R; Archelline 28; Azo- 

Bordeaux; Acid Bordeaux) 3.83 0.19 1% aq. C. 

252 Brilliant crocein 5.04 0.06 

1239 Carmine 0.3 • N.B. 

29 Chromotrope 2R (Chromotrope N2R; 
Chromotrope blue 2R; XL Car- 
moisine 6R; Fast fuchsin G; 
Acid phloxine GR) 
21 Chrysoidin R (Cotton orange; 

Cerotin orange) 
20 Chrysoidin Y (Brown salt R; 
Dark brown salt R) 
370 Congo red (Congo; Cotton red 8 or C; 

Direct red C, R or Y) 
--- Cresyl violet (Cresylecht violet 
cresyl fast violet ) (Nat . An. Co . . 

89 Crystal ponceau 6R (Ponceau 6R) 
681 Crystal violet (chloride) 

(Violet C,G or 78; Hexamethyl 
violet; Methyl violet 108; Gen- 
tian violet) 1.68 13.87 1% aq. N,V. 

Crystal violet Ciodide) 0.035 1.78 N.V. 

Crystal violet (chloride) 

resorcin ad. prod. 0.28 13.84 N,V. 

Crystal violet (chloride) 

hydroquin. ad. prod. 0.30 8.39 N,V. 

Crystal violet (chloride) 

pyrocatechin ad. prod. 0,79 24.87 N.V. 

715 Cyanol extra 1.38 0.44 P. 



-22- 



19.30 


0.17 


0.23 


0,99 


0.86 


2.21 


c, 


0.19 


r 
0,38 


0.25 


0.80 


0.06 



P. 


C 


• 


N. 






N. 


B. 


V. 


c, 


8. 


I. 


N, 


c, 


B. 


F. 







Coior Strength 

Index Name of Dye Water 95 % Solution Use 

Nu'^ber Alcohol Used 



771 Eosin B (Na salt) 

(Eosin BN.BA.BW.DHV; Saff rosin; 
Eosin scarlet; Scarlet J,JJ,V; 
Nopalin G; Imperial red; Eosin 

scarlet B) 39.11 0.75 C. 

768 Eosin Y (Na salt) 

(Eosin, water soluble; Bromo acid 
J.TS.XL or XX; Bromo fluorocein; 

Bronze bromo ES) 44.20 2.18 0.5% aq. C. 

Eosin Y (Mg salt) 1.43 0.28 C. 

Eosin Y (Ca salt) 0.24 0.09 C. 

Eosin Y (Ba salt) 0.18 0.06 C. 

130 Erika B 0.64 0.17 

254 Erythrin X 6.41 0.06 

773 Erythrosin (Mg salt) 0.38 0.52 C. 

Erythrosin (Ca salt) 0.15 0.35 C. 

Erythrosin (Ba salt) 0.17 0.04 C. 

Erythrosin, bluish (Na salt) 

(Erythrosin B,N, or JN; Pyrosin 

B; Eosin J; lodeosin; Dianthine B)11.10 1.87 1% aq. C,I. 
770 Ethyl eosin (Eosin, alcohol soluble; 

Eosin S) 0.03 1.13 0,5% al. C. 

Fast green FCF 16.04 0.35 C. 

176 Fast red A (Fast red AV,AL«BX,S or 
0; Cerasin; Rubidin; Cardinal 
Red; Roccellin) 1.67 0.42 

16 Fast yellow (Acid yellow; Fast 

yellow FY,G,S,BG; 18.40 0.24 C. 

766 Fluorescein (color acid) 0.03 2.21 C. 

Fluorescein (Na salt ) (Uranin) 50.20 7.19 C. 

Fluorescein (Mg salt) 4.51 0.35 C. 

Fluorescein (Ca salt) 1.13 0.41 C. 

Fluorescein (Ba salt) 6.54 0.56 C. 

677 Fuchsin, basic (Fuchsin RFN;Magenta; 

Basic rubin; Anilin) 0.30 10.00 N. 

676 Par arosanilin (chloride) 

(Basic rubin; Parafuchsin; 

Paramagenta) 0.26 5.93 N. 

Pararosani lin (acetate) 4.15 13.63 N. 

Rosanilin (chloride ) (Magental ) 0.93 8.16 N. 

692 Fuchsin, acid (Fuchsin S,SN,SS,ST, 

or S III; Acid magenta; Acid 

rubin) 12.00 0.3 C. 

678 New fuchsin (chloride )( Isorubin; 

Fuchsin NB; Magenta III) 1.13 3.20 N. 

Gentian violet (Methyl violet 2B) 1.50 3.00 1% al . N. 
666 Guinea green B 28.40 7.30 

1180 Indigo carmine (Indigotine la) 1.68 0,01 C. 

133 Janus green B (Diazin green S; 

Union green B) 5.18 1.12 0.1-1% aq. V,N,B. 



•23- 



Color 

Index 

Number 



Name of Dye 



Strength 
Water 95 % Solution 
Alcohol Used 



Use 



670 



657 



138 

684 
142 

680 
922 



924 

10 
152 
825 



826 
927 

728 



520 



914 

73 

150 



Light green SF yellowish 

(Light green 2G,S,2GN; Fast aci 
green N; Acid green) 

Malacliite green (oxalate) 

(Victoria green; New Victoria 
green extra, 0,1, or IIjDiamond 
green B,BX, or P extra; Solid green 



20.35 0.82 0.5% al. 



0; Light green N) 
Martius yellow, Na salt 

(Naphthol yellow; Manchester 

yellow ) 
Martius yellow, Ca salt 
Metanil yellow 

(Orange MNO or MN; Acid yellow R; 

Soluble yellow OL; Yellow M; 

Tropaeolin G) 
Methyl green 

(Double green SF; Light green) 

Methyl orange 

(Orange III; Helianthin; Gold 

orange MP; Tropaeolin D) 
Methyl orange (acid) 

Methyl violet (Gentian violet) 

(Dahlia B; Paris violet; Pyok- 

taninum coeraleum) 
Methylene blue (chloride) 

(Swiss blue 
Methylene blue (ZnCl2 double salt 
Methylene blue (iodide) 
Methylene green 
Naphtliol yellow G 
Narcein 
Neutral red (cliloride) 

(Toluylene red) 
Neutral red (iodide) 
Neutral violet 
New methylene blue N 

(Methylene blue NN) 
New Victoria blue R 

(New Victoria blue B or R; 

Corn blue B 
Niagara blue 4B 

(Pontamine sky blue 5BX; Direct 

sky blue; Benzo sky blue) 
Nile blue 2B 
Oil red 

(Confused with Sudanll) 
Orange I 

(Naphthol orange; Tropaeolin 

G,000 No.l) 



• 24- 



7.60 7.52 



4.57 
0.05 



0.16 
1.90 



5. 


36 


1.45 


7 


00 


0.25 






52 
.015 


0.08 
0.015 



1% aq. 



2.93 


15.21 


3.55 


1.48 


2.75 


0.05 


0.09 


0.13 


1.46 


0.12 


8.96 


0.025 


10.02 


0.06 


5.64 


2.45 


0.15 


0.16 


3.27 


2.22 



13.32 1.65 



0.54 3.98 



13.51 
0.16 

Nil 



Nil 
0.62 

0.39 



5.17 0.64 



1% aq. 



C. 



c. 

c. 
9- 

c. 

N. 



I,C. 

I.e. 



0.5-2% aq. N,V. 

N.V.I. 
N,V,I. 
N.V,I. 

N.V. 
C. 

c. 



V,N.C,I. 
V.N,C,I. 

I.N. 

N. 



V.N. 



F. 



C, I. 



I 



Color 

Index 

Number 



Name of Dye 



Strength 
Water 95 % Solution 
Alcohol Used 



Use 



151 Orange II 

(Gold orange; Orange A.P.R; Acid 

orange II, Y or A; Orange extra; 

Mandarin G; Tropaeolin 000 No. 2)11. 37 0.15 

27 Orange G 

(Wool orange 2G; Crystal orange 
GG) 10.86 0.22 

143 Orange IV 

(Orange N; Acid yellow D; 

Tropaeolin 00) 0. 16 0.20 

676 Pararosanilin (chloride) 

(Magenta 0; Basic rubin; Para- 
fuchsin; Paramagenta) 

Pararosanilin (acetate) 

714 Patent blue A 

Phenolphthalein 
774 Phloxine (Na salt) 

(Erythrosin BB or B extra; New 
pink) 
Phloxine (Mg salt) 
Phloxine (Ca salt) 
Phloxine (Ba salt) 
7 Picric acid 

28 Ponceau 2G 
186 Ponceau 6R 

Purpurine 4B 
741 Pyronin B (iodide) 
739 Pyronin Y 

(Pyronin G) 
148 Resorcin yellow 

749 Rhodamine B 

(Rhodamine 0; Brilliant 
pink B) 

750 Rhodamine G 
176 Roccellin 

(Fast red A.AV.AL.BX.S or 0; 
Cerasin; Rubidin; Cardinal red) 
Rosanilin (Chloride (Magenta I) 
779 Rose bengal (Na salt) 
Rose bengal (Mg salt) 
Rose bengal (Ca salt) 
Rose bengal (Ba salt) 
841 Safranin 

(Gossypimine ; Cotton red; 
Safranin Y or A) 
689 Spirit blue 

(Anilin blue, alcohol soluble; 

Gentiana blue 6B; Light blue; 

Lyon blue; Paris blue) Nil 



0.26 


5.93 


4.15 


13.63 


8.40 


5.23 


0.04 


10.00 


50.90 


9.02 


20.84 


29.10 


3.57 


0.45 


6.01 


1.17 


1.18 


8.96 


1.75 


0.21 


12.98 


0.01 




0.13 


0.07 


1.08 


8.96 


0.60 


0.37 


0.19 


0.78 


1.47 


1.34 


6.31 


1.67 


0.42 


0.39 


8.16 


36.25 


7.53 


0.48 


1.59 


0.20 


0.07 


0.17 


0.05 



N. 
N. 
P. 
I. 



C. 
C. 

c. 
c. 
c. 
p. 
p. 

N. 
N. 

N. 
C. 



5.45 3.41 1% aq. 



1.10 0.3% aq. 



V.C. 
C. 



c. 

N. 

c. 
c. 
c. 
c. 



N.V. 



C,B. 



-25- 



Color 

Index 

Number 



Name of Dye 



Water 



95 7c 
Alcohol 



Strength 

Solution 

Used 



Use 



24 Sudan I 
73 Sudan II 

(Oil scarlet; Fast oil orange 

II; Red B; Fat ponceau; Orange 

RR) 
248 Sudan III 

(Sudan G; Tony red; Scarlet B, 

fat soluble; Fat ponceau G; Oil 

red AS,0,B or 3B; Cerasin red) 
258 Sudan IV 

(Scarlet red; Fat ponceau; 

Fat ponceau R or LB; Cero- 

tine ponceau SB; Oil red IV) 
920 Thionin 

(Lauth' s violet ) 
925 Toluidine blue 

(Methylene blue T50 or T extra) 
728 Victoria blue R 

(New Victoria blue B or R; 

Corn blue B) 
690 Victoria blue 4R 

(Fat blue 4R) 
569 Victoria green 3B 
8 Victoria yellow 



Nil 



Nil 



0.37 



0.39 



Nil 0.15 



F. 



F. 



Nil 


0.09 




F. 


0.25 


0.25 




N.V 


3.82 


0.57 


0.3-1% aq. 


N,V 


0.54 


3.98 




V.N 


3.23 


20.49 




N. 


0.04 


2.24 






1.66 


1.18 




C. 



•26- 



I 



CHAPTER V. 
STANDARD CHEMICAL SOLUTIONS. STOCK SOLUTIONS AND SOLUBILITIES 

Preparation of Standard Solutions: 

Reagent grade chemicals and freshly distilled water should be used 
for all solutions. Weighings should be carried out in the low humidity 
room, using calibrated weights. Calibrated glassware should be used in 
all volumetric operations. Standard solutions should be issued in clean, 
dry Pyrex bottles. Rubber stoppers (cleaned in hot sodium hydroxide so- 
lution, then washed and dried) should be used for alkali solutions; glass 
stoppered Pyrex bottles should be used for the other solutions. 

Standard solutions should be issued only by the member of tlie staff 
assigned to this work or by the person in charge. The accuracy of the 
solution should be indicated on the label of each container. When an 
accurately standardized solution is requested by an investigator, the 
staff member should find out whether extreme accuracy is needed. If not, 
an attempt should be made to convince the investigator that an approx- 
imate solution is adequate. 
A. Standard Acids and Bases: 

The base solutions are standardized with solid potassium acid 
phthalate while the acid solutions are standardized with the standard 
bases. Thus, potassium acid phthalate is the reference standard for 
all acid and base solutions and the best grade of this compound should 

be used. 

The potassium acid phthalate is dried at 110°- 115° for two or three 
hours and then stored in a desiccator in the low humidity room. Samples 
are weighed in the Erlenmeyer flasks to be used in the titrations, and 
the weighings are carried out in the low humidity room. 
Normal Sodium Hydroxide : 

Saturated sodium hydroxide solution should be prepared for use 
during the following year and stored in a paraffined bottle closed by 
a cleaned rubber stopper. The sodium carbonate precipitates, leaving a 
clear supernatant solution which should be pipetted or carefully decanted 

from the carbonate. 

For each liter of normal sodium hydroxide, dilute 60 ml. of the 
saturated sodium hydroxide solution to one liter with freshly boiled 
distilled water and store in a paraffined bottle protected with a soda- 
lime tube. Adjust as closely as possible to one normal by single runs 
against potassium acid phthalate. Finally, accurately standardize the 



•27- 



solution by at least six runs against 7-8 gram samples of potassium acid 
phthalate, titrating to a faint pink with phenolphthalein. 
Tenth Normal Sodium Hydroxide : 

The procedure is the same as for normal sodium hydroxide, except 
that 6 ml. of the saturated sodium hydroxide solution is used per liter 
of solution, and 0.8-0.9 gram samples of potassium acid phthalate are used. 
Normal Hydrochloric Acid : 

For each liter of normal hydrochloric acid, dilute 86 ml. of the 
concentrated acid, sp. gr. 1.18, to one liter with distilled water. Adjust 
as closely as possible to one normal by titration with the standardized 
one normal sodium hydroxide. Finally, accurately standardize the acid 
by at least six titrations with 40-45 ml. samples of the standard base, 
to a faint pink with phenolphthalein. 
Tenth Normal Hydrochloric Acid: 

The procedure is the same as for normal hydrochloric acid, except 
that 8.6 ml. of concentrated acid, sp. gr. 1.18, is used per liter of 
solution, and the standard tenth normal sodium hydroxide is used in the 
titrations . 
Normal Sulfuric Acid: 

For each liter of normal sulfuric acid, slowly add 27 ml. of con- 
centrated sulfuric acid, sp. gr. 1.84, to about 250 ml. of distilled 

water, allow to cool, and dilute to one liter. Proceed as with normal 

hydrochloric acid. 

Tenth Normal Sulfuric Acid : 

Proceed as for normal sulfuric acid, using 2.7 ml. of concentrated 
sulfuric acid, sp. gr. 1.84, per liter of solution. Standardize with 
tenth normal sodium hydroxide. 
Normal Acetic Acid: 



For each liter of normal acetic acid, dilute 60 ml. of glacial 
acetic acid to one liter with distilled water. Proceed as with normal 
hydrochloric acid. 
B. Other Standard Solutions : 
Tenth Normal Potassium lodate : 

Weigh out 3.5672 grams of dried potassium iodate and make up to 
one liter with distilled water. This solution keeps very well and need 
not be freshly prepared each year. 
Tenth Normal Sodium Thiosulfate : 

For each liter of solution, dissolve 24.8 grams of hydrated sodium 
thiosulfate, Na2S203» 5H2O, in freshly boiled disti lied ^ater , add 10 ml. 
of tenth normal sodium hydroxide, and dilute to one liter. To standardize 



•28- 



against potassium iodate, dissolve 1-2 grams of reagent grade potassium 
iodide in 200 ml. of water in a 500 ml . Er lenmeyer flask, add ten drops 
of concentrated hydrochloric acid and test for free iodine with starch. 
If no free iodine is present, add 25.00 ml. of the potassium iodate 
solution and titrate the liberated iodine with the thiosulfate immedi- 
ately. The thiosulfate is added until the solution is a pale yellow, 
then 2-3 ml. of starch solution are added and the titration continued 
until the solution is colorless. The average of several titrations 
which agree closely should be taken as the normality of the thiosulfate 
solution. 

The thiosulfate solution should be prepared a year in advance. 
After this time its concentration changes very little. 
Tenth Normal Potassium Permanganate: 

Dissolve 3.25 grams of potassium permanganate in one liter of 
distilled water and boil the solution for 10-15 minutes, let stand 
overnight and then filter through sintered glass or asbestos. Stand- 
ardize with tenth normal thiosulfate by measuring out 25.00 ml. of the 
permanganate solution into a 500 ml. Erlenmeyer flask containing 3 grams 
of potassium iodide and 5 ml. of concentrated hydrochloric acid dissolved 
in 50 ml. of water. Let stand in the dark for 5 minutes, dilute to about 
200 ml. with water and titrate with the thiosulfate, using starch as the 
indicator. 

The addition of 10 grams of potassium hydroxide per liter of the 
permanganate solution increases its stability. 
Tenth Normal Potassium Bichromate: 



Dissolve 4.90 grams of potassium bichromate in distilled water and 
make up to one liter. Standardize with tenth normal thiosulfate by mea- 
suring out 25.00 ml. of the bichromate solution into a 750 ml. Erlen- 
meyer flask containing 10 ml. of concentrated hydrochloric acid and 3 
grams of potassium iodide in 50 ml. of water. Let stand in the dark for 
5 minutes, dilute to about 400 ml. with water and titrate with the thio- 
sulfate, using starch as the indicator. 

Owing to the green color of the final solution, it is advisable to 
carry out the titration under artificial light. 
Normal Silver Nitrate : 

Weigh out 169.89 grams of silver nitrate, previously dried at 110 , 
dissolve in distilled water, make up to one liter and protect the solution 
from the light. This solution is usually adequate for the standardization 
of chloride solutions but it may be standardized by gravimetric determi- 
nation as silver chloride. When greater accuracy is needed, the dry crys- 



• 29- 



tals may be fused for 15 minutes at 220 -250 . 
C. Approximate Standards: 

Prepare at the beginning of the season about 14 liters of 1.0 N 
hydrochloric acid by diluting the acid according to the information in 
the table on the strengths of acids; and of 1.0 N sodium hydroxide by 
weighing out the proper amount of alkali and making up to volume. 
Other approximate standards are to be made as required and every effort 
should be made to issue the proper solution thereby saving the stocks 
of the specially accurate solutions when these are not required. 

STRENGTH OF STOCK ACIDS AND BASES 



I 



Substance Molecular Molarity Normality Specific Percent Grams/ ml. to make 
Weight Gravity Liter one 1. of IN 

Acids 



Ac e t i c 


60.05 


17.36 


17.36 


1.05 


99.8 


1048 


57 




Butyric 


88.06 


10.78 


10.78 


0.959 


98-100 


949 


93 




HCl 


36.47 


11.50 


11.50 


1.19 


37 


440 


83 




Lactic 


90.08 


11.25 


11.25 


1.21 


85-90 


1030 


87 




Nitric 


63.02 


15.82 


15.82 


1.42 


70.5 


1001 


63 




Phosphoric 


98.00 


14.75 


44.25(3H) 


1.70 


85 


1445 


23 


(3H) 


Sulfuric 


98.08 


18.01 


36.03 


1.84 


94 


1730 


28 




Bases 



















Ammonium 17.03 14.7 14.7 0.90 28(NH3) 252 67 
Hydroxide 

SOLUBILITY OF COMPOUNDS* 



Solubility is expressed in grams of solute per 100 ml. of solvent at 20 C. 
KEY: s - soluble sl.s - slightly soluble 

inf - infinitely soluble i - insoluble 

vs - very soluble 
Substance Formula 

Acid 



Boric H3BO3 

Citric H3CgH50-7«H20 

Oxalic (C00H)2*2H20 

Phosphomolybdic 20Mo03« 2H3P04' 48H2O 

Picric CgH2(OH)2(N02)3 

Tartaric C2H2 (0H)2 (C00H)2 

Trichloracetic CCI3COOH 

Aluminum Chloride AlClo 



•30- 



Mol. Wt. 


Sol. H2O 


Sol. Alcohol 


61.84 


5.15 


5.56 


210.11 


135 


116 


126.06 


10 


23.7 


3939.78 


0.4 


vs 


229.08 


1.0 


4.91 


150.07 


139 


19.85 


163.40 


120 


s 


133.34 


69.87 


100 



i 



Substance Formula 

Al urn 

Amm. Iron Fe2(S04)3 (NH4)2S04«24 HjO 

Potas. Chrom. Cr2 (S04)3K2S04' 24 HjO 

Potas.Alumin. AI2 (SO4 )3K2S04« 24 H2O 

Amidol diaminophenol HCl 

Ammonium 



Acetate 

Chloride 

Molybdate 

Nitrate 

Oxalate 

Sul fate 

Barium 

Chloride 



NH4C2H302 




NH4C1 




(NH4)2Mo04 




NH4NO3 




(NH4)2C204- 


H2O 


(NH4)2S04 




BaCl2«2H20 





Hydroxide BaCOlDg'BHgO 

Calcium 

Chloride CaCl2 

Chloride CaCl2-2H20 

Chloride CaCl2'6H20 

Carbon Tet, CCI4 

Chloroform CHCI3 

Chromium Oxide Cr03 

Copper 

Acetate (ic) Cu(C2H302 )2* HgO 

Chloride (ic) CuCl2*2H20 

Chloride (ous) CU2CI2 

Sulfate (ic) CUSO4 

Sulfate (ic) CUSO4.5H2O 

Dextrose (glucose )CgH^ 2^6* ^2^ 

Ether C2H5OC2H5 
Glycine ' CH2NH2COOH 

Glycylglycine NH2CH2CONHCH2COOH 

Hydroquinone CgH4(0H)2 

Iodine I2 
Iron 



Chloride (ic) FeCl3'6H20 

Sulfate (ous) FeS04'7H20 

Lactose C^ 2^22^1 1* ^^'2^ 
Lead 



Mol. Wt. 


Sol. HjO 


Sol. Alco 


964.40 


124 


i 


006.51 


20 


i 


948.77 


11.4 


i 


197.01 


20.5 


sl.s 


77.06 


148 


V . s. 


53.50 


38 


0.6 


196.03 


3.5 


i 


80.05 


120 


3.8 


142.12 


4 


i 


132.14 


75 


i 


244.32 


35.7 


i 


315.51 


3.5 


si . s 


110.98 


74.5 


s 


147.03 


100 


s 


219.09 


300 


s 


153.84 


0.08 


inf. 


119.39 


1.0 


inf. 


100.01 


170 


s 


199.63 


7.2 


7.14 


170.52 


126 


s 


198.05 


0.0062 


i 


159.63 


40 


i 


249.71 


60 


i 


198.14 


82 


2 


74.10 


7.5 


inf. 


75.04 


25 


0.043 


132.12 


19.8 


sl.s 


110.08 


6.1 


V . s . 


253.84 


0.029 


20.5 


270.31 


300 


s 


278.02 


28 


i 


360.31 


17 


i 



•31- 



Substance 


Formula 


Acetate 


Pb(C2H302)2-3H20 


Chloride 


PbCl2 


Lithium 




Carbonate 


Li 2CO3 


Chloride 


LiCl 


Magnesium 




Chloride 


MgCl2'6H20 


Sulfate 


MgS04'7H20 


Maltose 


12^221 1 * 2 


Manganese 




Chloride 


MnCl2»4H20 


Mercuric 




Chloride 


HgCl2 


Osmium 




Tetroxide 


OSO4 


Potassium 




Acetate 


KC2H3O2 


Bromide 


KBr 


Carbonate 


K2CO3 


Bicarbonate 


KHCO3 


Chloride 


KCl 


Cyanide 


KCN 


Bichromate 


K2Cr207 


Ferricyanide 


K3Fe(CN)g 


Ferrocyanide 


K4Fe(CN)g-3H20 


Hydroxide 


KOH 


Iodide 


KI 


Nitrate 


KNO3 


Oxalate 


K2C2O4.H2O 


Permanganate 


KMnO^ 


Phosphate (mono) 


KH2PO4 


Phosphate (di) 


K2HPO4 


Acid Phthalate 


KHCgH^O^ 


Sulfate 


K2SO4 


Thiocyanate 


KCNS 


Silver Nitrate 


AgN03 


Sodium 




Acetate 


NaC2H302-3H20 


Borate , te tra 


Na2B4O-7«10H2O 


Carbonate 


Na2C03 



Mol, Wt. 


Sol. 


H2O 


Sol. 


Alcohol 


379.30 


50 








i 


278.11 


1. 









i 


73.88 


1. 


33 






i 


42.40 


75 








2.48 


203.33 


167 








50 


246.50 


71 








s 


360.31 


100 




V. 


si 


. s. 



197.91 



271.52 



254.80 



225 



6.9 



33 



98.12 


253 


33 


119.01 


63 


0.5 


138.19 


112 


i 


100.10 


27 


i 


74.55 


34.7 


i 


65.10 


50 


s 


294.21 


14 


i 


329.18 


40 


i 


422.32 


30 


i 


56.10 


110 


V. s. 


166.03 


140 


14.3 


101.10 


31.6 


i 


184.21 


33 


i 


158.03 


10 


i 


136.14 


33 


i 


174.22 


V . s . 


V. s . 


204.22 


10 


i 


174.26 


11.7 


i 


97.17 


185 


s 


169.89 


288 


V. si . s_. 


136.06 


100 


2.18 


381.43 


7 


V. s 1 . s . 


105.99 


24 


s 1 . s . 



•32- 



Substance 




Formula 


Carbonate 




Na2CO3«10H2O 


Bicarbonate 


NaliC03 


Oi loride 




NaCl 


Chromat e 




NagCrO^.lOHgO 


Citrate 




Na3CgH50-7'2H20 


Cyanide 




NaCN 


Hydroxide 




NaOH 


Ni trat e 




NaN03 


Oxalate 




Na2C204 


Phosphate 


(mono) 


NaH2P04.H20 


Phosphate 


(di) 


Na2HP04.12H20 


Phosphate 


(di) 


Na2HP04.2H20 


K Tartrate 




NaKC4H40g.4H20 


Sulfate 




Na2S04 


Sulfate 




Na2SO4.10H2O 


Sulfite 




NajSOg 


Bisulfite 




NaHS03 


Thiocyanate 


NaCNS 


Thiosul fat 


e 


Na2S203.5H20 


Veronal 




NaC8H,,N203 


Sucrose 




C,2H220ii 


Thymol 




CgH3(CH3)(0H)(CgH7) 


Urea 




C0(NH2)2 


Uranium Ni 


trate 


U02(N03)2*6H20 


Urethane 






Ethyl 




NH2COOC2H5 


Phenyl 




CgH5NHCOOC2H5 


Zinc 







Mol. Wt. Sol. 



ZnCl2 



ZnS04«7H20 



Chloride 
Sulfate 

*For additional information cf. Handbook 
Chemical Dictionary; Merck's Index; Seidell's 
and Organic Substances. 

1. Forms the acid in water. 



286.15 

84.01 

58.46 

342.16 

294.10 

49.01 

40.01 

85.01 

134.01 

138 

358.24 
178.05 
282.19 
142.06 
322.22 
126.06 
104.07 
81.07 
248.20 
206.18 
342.30 
150.21 
60.05 
502.18 

89.08 
165.19 

136.29 
287.56 



65 

10 

36. 

60 

50 

50 

103 
93 
3. 

110 
17 
50 
60 
14 
35 
26 
25 

139 

250 
20 

200 
0. 

100 

200 

100 
si. s. 

430 
96 



H2O Sol. Alcohol 



si . s 
si . s 
si . s 
si. s 
si. s 

V. s 

si. s 



09 



V. si , 



si . s 



V. s 

si. s . 

0.9 

350 

15.8 



166 
V. s. 

100 
si. s . 



of Chemistry and Physics; 
Solubilities of Inorganic 



■33- 



GRAVIMETRIC FACTORS 

A gravimetric factor is a stoichiometric ratio between the weights of 
two substances. In tlie case of Table A, it is the ratio of the molecular 
weights of the hydrated/anhydrous salts. To obtain the weight of hydrate 
equivalent to a given- amount of anhydrous salt, multiply the weight of 
anhydrous salt by the factor. In the case of Table B, the factor is the 
ratio of equivalent weights as shown in the formula fraction. To obtain 
tlie weight of substance in the numerator equivalent to a known weight of 
substance in the denominator, multiply the latter weight by the factor. 
TABLE A: Hydrated : Anhydrous Salts 

Factor 

1.095 

1.401 

1.145 

1.974 

1.325 

1.440 

1.141 

1.264 

1.268 

1.287 

1.564 

1.100 

1.667 

1.568 

1.165 

1.164 

1.587 

2.135 

2.048 

1.572 

1.477 

1.119 

1.147 

1.659 

1.170 

1.486 

1. 140 

1. 176 

1.150 



-34- 



Cation 


Formula of Hydrate 


Acids 


H3Citric+H20 




H2C204+2H20 


Ammonium 


(NH4)2C204*H20 


Calcium 


CaCl2+6H20 




CaCl2*2H20 




Ca(N03)2*4H20 




CaC204*H20 




CaS04i-2H20 


Copper 


CuCl2*2H20 




Cu(N03)2+3H20 




CuS04*5H20 


Dextrose 


C6H,206*H20 


Iron 


FeCl3+6H20 




FeCl2+4H20 


Lead 


Pb(OAc)2+3H20 


Lithium 


Li2S04+H20 


Magnesium 


MgBr2+6H20 




MgCl2-^6H20 




MgS04*7H20 


Manganese 


MnCl2+4H20 




MnS04+4H20 




MnS04+H20 


Potassium 


K4Fe(CN)g+3H20 


Sodium 


NaOAc+3H20 




Na2C03+H20 




Na2Cr04+4H20 




Na3Citric+2H20 




Na2Mo04+2H20 




NaH2P04*H20 



1 



Cation Fo 


rmul 


a of Hydrate 


Factor 




Na 


2HPO 


4+12H20 


2.522 




Na 


3PO4 


+I2H2O 


2.319 




Na 


2SO4 


+ IOH2O 


2.269 




Na 


2S+9H2O 


3.078 




Na 


2SO3 


+ 7H2O 


2.001 




Na 


2S2O 


3*5H20 


1.560 




Strontium Sr 


CI2 + 


6H2O 


1.682 




Zinc ZnSO^+ 


7II2O 


1.781 




TABLE B: SUBSTITUTION 


OF IONS 






Ions Exchanged 




Formula Fraction 




Factor 


Sodium-»Potassium 




KHC03/NaHC03 

K2HP04/Na2HP04 

NaH2P04+H20/KH2P04 




1.192 
1.227 
1.014 


Strontium-»Calcium 




. CaCl2+2H20/SrCl2 




0.927 


Chloride*Bromide 




NaBr/NaCl 
KBr/KCl 




1.761 
1.576 






CaBr2+2H20/CaCl2*2H 


2O 


1.604 






MgBr2+6H20/MgCl2+6H 


2O 


1.437 


Chloride-Iodide 




Nal/NaCl 
KI/KCl 




2.564 
2.228 






Cal2+6H20/CaCl2+2H2 





2.732 






Mgl2+8H20/MgCl2+6H2 





2.076 


Chloride-»Nitrate 




NaN03/NaCl 
KNO3/KCI 




1.454 
1.356 






Ca(N03)2+4H20/CaCl2 


•►2H2O 


1.605 






Mg(N03)2+6H20/MgCl2 


+ 6H2O 


1.261 



-35- 



CHAPTER VI. 

INDICATORS AND INDICATOR SOLUTIONS 
Revised 1951 by J. D. Ostrow 
Reference: Clark, W. M. , The Determination of Hydrogen Ions. (3rd Ed) 

INDICATOR SOLUTIONS: 

In the colorimetric determination of pH's, aqueous solutions con- 
taining 0.04% of the indicator are generally used. However, to simplify 
preparation and increase stability, stock solutions of 0.4% concentration 
are first prepared, and the test solutions made up as needed by dilutions 
of the stock. Since the indicators, as such, are insoluble in water, they 
are first converted to their monosodium salts, prior to dissolution in the 
specified volume of water. 

To prepare the 0.4% stock solutions: weigh exactly 1.00 g. of the 
indicator into an agate mortar. Add the number of ml. of 0.05N NaOH 
specified in the table below, and grind with an agate pestle until dis- 
solved. Transfer quantitatively, with rinsing, to a 250 ml. volumetric 
flask, and dilute to the mark with distilled water. 

To prepare the 0.04% test solutions: dilute 1 part of the stock 
solution with 9 parts of distilled water, using accurately calibrated 
pipettes and flasks. 

INDICATOR SETS: 

For determination of pH' s to the nearest 0.1 pH unit, sets of indi- 
cator color standards are used. Each set covers the detectable range of 
color change of a given indicator, as listed in the table below, and pro- 
ceeds in steps of 0.2 pH units, intermediate values being estimated visually. 

Each set consists of nine colorimetr ical ly matched 15 X 150 mm. test 
tubes, each calibrated at 10 ml. If the tubes cannot be matched on a 
colorimeter, the following method will suffice: all tubes whose 10 ml. 
graduation falls at the same height above the base of the tube will have 
the same inside diameter, and therefore be color imetr ical ly identical, 
except for variations in the thickness of the walls of the tubes. They 
may, therefore, be used in the same indicator set, with little resultant 
error . 

Each tube is filled to the mark with buffer solution at the proper 
pll, tlie bottom of the meniscus being read in each case. Five drops, or 
0.25 ml., of the 0.04% indicator test solution is then added. The tube. 



•37- 



is stoppered with a cork which is protected from the solution by cellophane, 
and then inverted several times until thoroughly mixed. It is then labelled 
with the name of the indicator, the volume, and the concentration of the 
indicator solution used, and the pH of the buffer. Tubes must be freshly 
prepared each summer, after thorough washing, though labels may be reused 
without removal if they have been protected by Scotch Tape or label varnish. 
Stability is also enhanced by storage in the dark, but in any case, the 
sets are good only for three months at the most. 

A set is issued in a test tube rack with an empty, matched tube and 
a dropping bottle of the indicator solution. To avoid errors in the volume 
of indicator used, the same bottle should be used in making the standard 
tubes, since this will insure that the drops are all from the same dropper 
and, therefore, of identical size. 

To use the sets, the proper indicator is first determined by adding 
a drop of various indicators to small amounts of the unknown. The empty 
tube of the proper set is then filled to the mark with the unknown solu- 
tion, and five drops of the indicator solution added. After thorough mix- 
ing, the color of the unknown tube is matched with those of the standards, 
using a comparator block. Note that the most commonly used indicator is 

Phenol Red, since it exhibits its maximum color gradation in the range of 
physiological pH's. 

TABLE OF INDICATORS 

In each case, add 0.25 ml. (5 drops) of 0.04% indicator solution to 
10 ml. of the solution to be tested. 



Indicator 

*Thymol Blue 

Brom Phenol Blue 

Brom Cresol Green 

Chlor Phenol Red 

Brom Cresol Purple 
Brom Thymol Blue 

Phenol Red 

Cresol Red 

Meta Cresol Purple 

Thymol Blue 



pH Range 

1.2-2.8 

3.0-4.6 

3.8-5.4 

4.8-6.4 

5.2-6.8 
6.0-7.6 

6.8-8.4 

7.2-8.8 

7.4-9.0 

8.0-9.6 



ml. of 0.05N 
NaOH per gram dye 

43 
30 
29 

47 
37 



Color Change 

Red — Yellow 
Yellow— Blue Violet, 
Yellow — Blue 
Yellow — Red 
Yellow — Violet 



32 Yellow - Blue 

57 Yellow — Red 

5 3 Yellow - Red 

53 Yellow — Purple 

43 Yellow — Blue 

*Thymol Blue has two ranges, one acid and one alkaline. In the acid 

range, use twice as much indicator as usual, that is, 10 drops (0.5 ml. ) 
of 0.04% solution per 10 ml. of unknown. 



38- 



aiAPTER VII. 
BUFFERS 

Each taole here given is for preparing a given volume of buffer, con- 
taining a specified concentration of the desired buffer anion, at the various 
pH's listed. These volumes and concentrations are given above each table. 
For each specie of buffer, two solutions are needed; a standard stock solu- 
tion of some salt of the buffer anion, and a solution of either standard 
acid or alkali, usually NaOH or HCl . Instructions for making up the stock 
solutions are given above each table of buffers. A given volume of this 
stock is pipetted into a flask, the required amount of standard acid or 
alkali buretted into the same flask, and this mixture then diluted to the 
proper volume with distilled water, using a volumetric flask. This will 
give the listed volume of buffer at the specified anion concentration and 
at exactly the pH listed. If smaller or larger volumes are desired, the 
amounts of stock solution and standard alkali or acid should be altered 
proportionally. If an anion concentration other than that specified is 
desired, one alters the amounts of stock solution and standard acid or 
base proportionally, but does not change the total volume upon dilution. 
This will not significantly alter the pH, provided that the anion con- 
centration is somewhere between 1/4 X and 2X that specified in the table. 
Within this range, the effects of dilution on the pH of buffers is negli- 
gible. In all cases, remember that the important factor is the ratio of 
stock anion solution to standard acid or base solution. As long as one 
maintains the proportions of these two solutions to each other, the buffer 
will have the proper pH. 

In making up the stock anion solutions, it is always necessary to use 
the Merck Reagent Grade or Baker's Analyzed Grade chemical. If these 
bottles are initially opened in the dry room and kept in that room at low 
humidity at all times, the chemicals may generally be used directly with- 
out drying. For high accuracy, however, (i.e. in making standard buffers), 
the salts must be treated as directed and dried in an oven. 



39- 



I 



POTASSIUM CHLORIDE BUFFER: pH 1.0 - 2.2 

Ref: Clark and Lubs: J. Biol. Chem; 2S 479 (1916) 

Stock Solution: l.OOOM Potassium Chloride 

Prepared by dissolving 14.912 g. of KCl in enough distilled water to 
make 200 ml. of solution. Merck's Reagent may be used directly if freshly 
opened in the dry room and kept there. For greater accuracy, dry four 
hours at 120*'C. 

Buffers : 



pH 


ml. l.OOOM HCl 


1.0 


19.40 


1.2 


12.90 


1.4 


8.30 


1.6 


5.26 



To make 200 ml. of 0.05 M (1/20 M) buffer, pipette 10 ml. of the 
1.000 M stock solution into a 200 ml. volumetric flask, add the volume 
standard (l.OOOM or 0. lOOOM as specified) HCl listed below, and dilute 
to the mark with distilled water. 

pH ml. O.IOOOM HCl 

1.8 32.20 

2.0 21.20 

2.2 13.40 

POTASSIUM HYDROGEN PHTHALATE BUFFERS 

With HCl: pH 2.2 - 3.8 With NaOH: pH 4.0 - 6.2 

Ref: Clark and Lubs: J. Biol. Chem. ^ 479 (1916) 
Stock Solution : 0.200M Potassium Biphthalate 

Prepared by dissolving 40.828 g. of Merck's Reagent Potassium 
Biphthalate in enough distilled water to make 1 liter. The Merck 
product may be used directly if opened and kept in the dry room. For 
greater accuracy, dry for several hours at 110 -120 C 

Buffers : 



To prepare 200 ml. of 0.05 M Phthalate buffer, pipette 50 ml. of 
the stock 0.200M solution into a 200 ml. volumetric flask, add the spec- 
ified amount of the standard HCl or NaOH (l.OOOM or O.IOOOM as listed) 
and dilute to the mark with distilled water. 



pH 


ml . 


of l.OOOM HCl 


2.2 




9.34 


2.4 




7.92 


2.6 




6.59 


2.8 




5.28 



pH 


ml. of O.IOOOM HCl 


3.0 


40.64 


3.2 


29.40 


3.4 


19.80 


3.6 


11.94 


3.8 


5.26 



-40- 



1 



pH 


ml. 


of 


0. lOOOM NaOH 






pH 


ml . of 


l.OOOM NaOH 


4.0 






0.80 










5.2 






5.99 


4.2 






7.40 










5'. 4 






7.09 


4.4 






15.00 










5.6 






7.97 


4.6 






24.30 










5.8 






8.60 


4.8 






35.40 










6.0 






9.09 


5.0 






47.70 










6.2 






9.40 












CITRATE 


BUFFERS 


















pH 2. 


2 


- 6. 











Reference 


Reca 


leu 


lated f 


rom 


Koltho 


ff 


and 


Vleeschhouwer; 






Bioc 


hem 


. Zeit. 


18; 


, 144 


(1927) 


179, 


410 


(1926) 




Stock Soli 


Jtion: 


0. 


500M Ci 


trie 


Acid 















Weigh out 105.055 g. of dry, crystalline Citric Acid (CoHgCU»H20) 
and dissolve in enough water to make 1 liter. Merck Reagent grade should 
be used, and it is satisfactory to use a bottle opened and kept in the 
dry room. To be sure of the molarity, titrate with l.OOOM NaOH. The 
end-point is a distinct red color of the phenolphthalein indicator. 

Bu f f e r s : 



To make 250 ml. of . I M Citrate Buffer, pipette 50 ml. of the 
0.500M stock into a 250 ml. volumetric flask. Add the number of ml. of 
l.OOON NaOH indicated below and dilute to the mark with distilled water, 



pH ml. l.OOONaOH 

2.2 2.23 

2.4 4.63 

2.6 7.13 

2.8 10.10 

3.0 13.40 

3.2 17.10 

3.4 20,50 

3.6 23.93 

3.8 26.00 

4.0 29.50 



pH 


ml. l.OOON Na 


4.2 


33.15 


4.4 


36.85 


4.6 


40.75 


4.8 


44.60 


5.0 


48.35 


5.2 


52.10 


5.4 


55.50 


5.6 


59.00 


5.8 


62.20 


6.0 


65.60 



■41- 



ACETATE BUFFERS 

pH 3.6 - 5.6 

Reference: Recalculated from Walpole, J. S. , J. Chem. Soc. 

105 2501 (1914) 
Stock Solutions: l.OOON Acetic Acid 

l.OOON Sodium Hydroxide NaOH 
Both of these solutions are prepared according to the section on 
standard solutions ( CHAPTER V ). 

Buffers: 



To make 1 liter of O.IM Acetate Buffer, pipette 100 ml. of the l.OOON 
Acetic Acid stock solution into a 1 liter volumetric flask, burette in the 
specified amount of l.OOON NaOH, and dilute to the mark with distilled water. 



pH 


ml. 1.00 


3.6 


7.5 


3.8 


12.0 


4.0 


18.0 


4.2 


26.5 


4.4 


37.0 


4.6 


49.0 



pH 


ml. 1.000 


4.8 


60.0 


5.0 


70.5 


5.2 


79.0 


5.4 


85.5 


5.6 


90.5 



PHOSPHATE BUFFERS 

pH 5.8 - 8.0 

Reference: Recalculated from Clark and Lubs; J. Biol. Chem. 
^ 479 (1916) 

Stock Solution: l.OOOM Potassium Phosphate, Monobasic 

(Potassium Dihydrogen Phosphate KH2P0«) 

Prepared by dissolving 136.14 g. of KHpPO^ in enough distilled water 
to make 1 liter of solution. Merck's anhydrous reagent may be used directly 
if freshly opened in the dry room and kept there. For greater accuracy, dry 
for two hours at 120 C and place in a desiccator. The solid is conveniently 
weighed in 25 - 30 g. lots, using glazed weighing paper. 

Buffers : 



To make 1 liter of O.IM Phosphate Buffer pipette 100 ml. of the l.OOOM 
Phosphate stock solution into a 1 liter volumetric flask, add the volume of 
l.OOON NaOH specified below, and dilute to the mark with distilled water. 



•42- 



pH 


ml. 1.000 


5.8 


7.32 


6.0 


11.28 


6.2 


17.10 


6.4 


25.20 


6.6 


35.48 


6.8 


47.20 



pH 


ml. l.OOON NaOH 


7.0 


59.08 


7.2 


69.80 


7.4 


78.68 


7.6 


85.48 


7.8 


90.34 


8.0 


93.70 



PHOSPHATE BUFFERS 

pH 5.8 - 8.0 

An alternate method of preparing phosphate buffers is to weigh out 
the proper proportion of dibasic and monobasic phosphate salts and dissolve 
to volume with distilled water. This procedure is of advantage under two 
circumstances : 

a) When buffers in pyrex distilled water are desired, since this 
avoids the time-consuming procedure of preparing standard 
NaOH in pyrex distilled water. 

b) When pure potassium or pure sodium phosphate buffers are 
desired, without the presence of the other ions. The advan- 
tage of the solids method is that no new standard solutions need 
be prepared. 

To make 1 liter of M/15 Phosphate Buffer, weigh accurately the amount 
of monobasic phosphate (Na or K as desired) and the amount of dibasic phos- 
phate (Na or K as desired) into a 1 liter volumetric flask, dissolve, and 
dilute to the mark with distilled water. Since dissolution may be slow, 
it is best, at first, to add 700 ml. of hot distilled water and shake until 
dissolved, then cool to room temperature and dilute to volume. 

In each case, use Merck reagent grade salts of the formulas given 
below. Freshly opened chemicals, kept in the dry room, may be used with- 
out other drying. 

PHOSPHATE BUFFERS M/15 
Reference: Recalculated from Sorenson, S.P.L. ; Ergeb. Physiol. 
22_393 (1912) 
Monobasic Salts Dibasic Salts 

KH2PO4 NaHgPO^'HjO pH K2HPO4 Na2HP04 

8.327g. 8.442g. 5.8 0.9580g. 0.7809g. 

7.942 8.051 6.0 1.452 1.183 

7.442 7.545 6.2 2.090 1.704 



-43- 



6.648 


6.739 


5.695 


5.773 


4.583 


4.646 


3.540 


3.588 


2.587 


2.622 


1.770 


1.794 


1.248 


1.265 


0.7942 


0.8051 


0.4992 


0.5060 



K2HPO4 


Na2HP04 


3.106 


2.532 


4.326 


3.526 


5.748 


4.685 


7.083 


5.774 


8.303 


7.768 


9.348 


7.620 


10.015 


8.164 


10.596 


8.637 


10.973 


8.945 



Monobasic Salts Dibasic Salts 

KH2PO4 NaH2PO4«H20 pH 

6.4 
6.6 
6.8 
7.0 

7.2 
7.4 
7.6 
7.8 
8.0 

Mix the monobasic and dibasic salts, dissolve in 700 ml. of hot 
distilled water, cool to room temperature, and dilute to 1 liter in a 
volumetric flask. 

BARBITAL (VERONAL) BUFFER 

pH 6.8 - 9.2 
Reference: Recalculated from J. B.C. 87 33 (1930) 

Stock Solution: 0.500M Sodium Veronal (also called Disodium Barbital, 
Barbital Soluble) 
Prepared by weighing out 51.545 g. of Veronal Sodium (Merck Powder) 
and dissolving in enough distilled water to make 500 ml. The powder, as 

it comes in the bottle, may be used directly without drying. 

] 
Buffers : 



To make 500 ml. of O.IM Veronal Buffer pipette 100 ml. of the 0.500M 
stock solution into a 500 ml. volumetric flask, add the volume of l.OOOM^HCl 
specified below, and dilute to the mark with distilled water. 

tpH ml. l.OOON HCl 

6.8 45.80 

7.0 43.30 

7.2 40.25 

7.4 36.05 

7.6 31.30 

7.8 25.53 

8.0 19.85 

■•"It is difficult to prepare buffers of a pH lower than 8.8. This is due 
to tlie ppt. of barbituric acid at low pHs . 

*IIere, for better accuracy, ten times as much O.IOOON HCl may be 
used instead. 



-44- 



pH 


ml. l.OOON HCl 


8.2 


15.05 


8.4 


10.75 


8.6 


7.40 


8.8 


5.07 


9.0 


3.42* 


9.2 


2.52* 






GLYCYLGLYCINE BUFFERS 

pH 7.6 - 9.0 

Reference: Determined experimentally in M.B.L. Chemical Room by 
J. D. Ostrow and J. B. Russell, 1951. Data available 
on request. 

Stock Solution : 0.500M Glycy Iglycine 

Weigh accurately 33.030 g. of glycylglycine into a 500 ml. volu- 
metric flask and dilute to the mark with distilled water. Use high purity 
chemical directly from a bottle opened and kept in the dry room. 

Buffers : 



To make 250 ml. of O.IM buffer pipette 50 ml. of the 0.500M stock 

solution into a 250 ml. volumetric flask. Add the volume of l.OOON 

NaOH indicated in the table below, and dilute to the mark with distilled 
water . 



pH 


ml. 


l.OOON NaOH 


7.6 




4.40 


7.8 




6.35 


8.0 




9.10 


8.2 




11.85 



pH 


ml. l.OOON NaOH 


8.4 


14.63 


8.6 


17.46 


8.8 


19.52 


9.0 


21.10 



Note: A charge will be made for all glycylglycine buffers due to the 
expense of this chemical. 

BORATE -KCl -NaOH BUFFERS 

pH 7.8 - 10.0 

Reference: Clark and Lubs; J. Biol. Chem. _25_ 479 (1916) 

Stock Solution : A mixture of 0.500M Boric Acid H3BO3 and 0.500M 
Potassium Chloride KCl 

Weigh out 31.0120 g. Boric Acid and 37.280 g. of KCl and dissolve in 
enough distilled water to make 1 liter of solution. Use Merck's Reagent 
grade chemicals. The solid may be weighed directly from the bottle, with- 
out previous drying, if freshly opened and kept in the dry room. For 
better accuracy, however, the KCl should be dried at 120 C in an oven for 
four hours, and the Boric Acid dried in thin layers over CaCl2in a desic- 
cator. Under no circumstances should the Boric Acid be heated above 50 C, 
or it loses water of constitution forming metaboric acid HBO2. 

Buffers : 

To make 1 liter of 0.0 5M buffer, pipette 100.0 ml. of the . 5M stock 
solution into a 1 liter volumetric flask, add the volume of l.OOON NaOH 
specified below, and dilute to the mark with distilled water. 



-45- 



pH 


ml. l.OOON 


NaOH 


pH 


7.8 


2.65 




9.0 


8.0 


4.00 




9.2 


8.2 


5.90 




9.4 


8.4 


8.55 




9.6 


8.6 


12.00 




9.8 


8.8 


16.40 




10.0 



ml. l.OOON NaOH 

21.40 
26.70 
32.00 
36.85 
40.80 
43.90 

CARBONATE-BICARBONATE BUFFERS 
pH 9.4 - 10.4 

Reference: Determined in M.B.L. Chemical Room by J. D. Ostrow and 
J. B. Russell, 1951. Data available on request. 

Stock Solution: 0.500M Sodium Bicarbonate 

Weigh accurately 21.0050 g. NaHCOg into a 500 ml. volumetric flask 

and dissolve to the mark with distilled water. Use Merck's reagent grade. 

The material may be used without drying if freshly opened in the dry room, 

but for more accuracy, dry at 110 C for four hours and store in a desiccator, 

Buffers : 

To make 250 ml. of O.IM Carbonate buffer, pipette 50ml. of the 0.500M 
Bicarbonate stock solution into a 250 ml. volumetric flask, burette in the 
volume of l.OOON NaOH indicated in the table below, and dilute to volume 
with distilled water. 

pH ml. l.OOON NaOH pH ml, 

9.4 4.85 10.0 

9.6 7.63 10.2 

9.8 10.70 10.4 



l.OOON NaOH 

13.73 
16.78 
19.58 



HIGH-pH PHOSPHATE BUFFER 
pH 11.0-12.0 

Reference: Biochem. Zeitschr. 189 191 (1927 ) ;Kolthof f and Vleeschhouwer 

Stock Solution: 0.500M Na2HP04 

Weigh out accurately 71.01 g. and dilute to 1 liter in a volumetric 
flask. Freshly opened reagent grade chemicals may be used if kept in the 
dry room. For greater accuracy, dry in an oven for two hours at 120 C. 

Bu f f e r s : 

To prepare 250 ml. of O.IM Phosphate buffer pipette 50 ml. of the 
0.500M stock secondary phosphate solution into a 250 ml. volumetric flask, 
add the volume of l.OOON NaOH indicated on the table below, and dilute to 
the mark with distilled water. 



-46- 



pH ml. l.OOON NaOH pH ml. l.OOON NaOH 

11.0 4.13 11.6 12.25 

11.2 6.00 11.8 16.65 

11.4 8.67 12.0 21.60 

McILVAINE BUFFERS 
pH 2.2 - 8.0 
Reference: J. Biol. Chem. _49_ 183 (1921) 

Stock Solutions : 

A) 0.500M Citric Acid: 

Weigh out 105.055 g. of dry Citric Acid (CgHgOy*H20) and dissolve 
in enough water to make 1 liter. The Merck reagent grade, if taken 
from a bottle freshly opened in the dry room, is satisfactory. To 
be sure of the molarity, standardize with l.OOOM NaOH. The titration 
is carried to a distinct red color of the phenolphthalein indicator. 

B) 0.500M Na2HP04: 

Weigh out 71.01 g. accurately and dilute to 1 liter with distilled 
water in a volumetric flask. Freshly opened Merck Reagent or Baker's 
analyzed grade chemicals may be used if kept in the dry room, but 
for greater accuracy, dry in an oven for two hours at 120 C and 
place in a desiccator. 

Buffers: 



To prepare 200 ml. of buffer, burette the amounts of stock solutions 
indicated below into a 200 ml. volumetric flask, and dilute to the mark 
with distilled water. 



pH 


ml. 0.500M NajHPO 


2.2 


1.60 


2.4 


4.96 


2.6 


8.72 


2.8 


12.68 


3.0 


16.44 


3.2 


19.76 


3.4 


22.80 


3.6 


25.95 


3.8 


28.40 


4.0 


30.84 


4.2 


33.12 



ml. 0.500M Citric Acid 

39.20 
37.52 
35.64 
33.66 
31.78 

30.12 
28.60 
27.12 
25.80 
24.58 
23.44 



-47- 



pH 


ml. 0.500M 


4.4 


35.28 


4.6 


37.40 


4.8 


39.44 


5.0 


41.20 


5.2 


42.88 


5.4 


44.60 


5.6 


46.40 


5.8 


48.36 


6.0 


50.52 


6.2 


52.88 


6.4 


55.40 


6.6 


58.20 


6.8 


61.80 


7.0 


65.88 


7.2 


69.56 


7.4 


72.68 


7.6 


74.92 


7.8 


76.60 


8.0 


77.80 



ml. 0.500M Citric Acid 

22.36 
21.30 
20.28 
19.40 
18.56 
17.70 
16.80 
15.82 
14.74 
13.56 
12.30 
10.90 

9.10 

7.06 

5.22 

3.66 

2.54 

1.70 

1.10 



UNIVERSAL BUFFER (TEORELL) 
pH 2.0 - 12.0 
Reference: Teorell, Tand and Stenhagen, Biochem. Zeitschr 



299 416-9 (1938) 



General : 



This buffer contains the buffer anions phosphate, borate, and citrate. 
It covers the pH' s 2 - 12, and has a practically linear buffer capacity in 

the range pH 3 - 11. Between these values, 5 ml. of O.IN acid or base 

shifts the pH about 1 unit. The molarities of the various ions in the 
final buffers are as follows: 

Phosphate - M/lOO Borate - M/lOO 

Citrate - M/150 Sodium - M/15 
Standard Solutions: 



1) l.OOON NaOH...See section on standards. 

2) O.IOOON HCl...See section on standards. 

3) IN Phosphoric Acid (H3PO4) ... Dilute 17.5 ml. of 85% highest grade 
H3PO4 to 500 ml. Standardize by titrating against 20 ml. aliquots of the 
standard NaOH, using phenolphthalein indicator. The end-point is taken 
as last trace of rose color, after the initial bright red has lightened 



-48- 



into the rose tint. Calculate the ml.'s of the acid equivalent to 100 ml. 
of the NaOH, 

4) IN Citric Acid. (H3CgH507* H2O) ... Dissolve 35 g. of the Merck reagent 
crystals in enough distilled water to make 500 ml. Standardize against the 
l.OOON NaOH exactly as with the phosphoric acid, taking the same end-point. 
Store in the refrigerator. 

5) Boric Acid Crystals. (H3BO3 ) . . . Dry Merck's reagent grade in thin 
layers over CaCl 2 in a desiccator. 

Stock Solution: 

Using a burette, measure into a 1 liter volumetric flask the volumes 
of phosphoric acid solution (3) and citric acid solution (4) equivalent 
to 100.0 ml. of the IN NaOH, as determined by the titrations. Add 343.0 
ml. of IN NaOH and 3.54 g. of the Boric Acid and dilute to the mark with 
distilled water. 

Buffers : 



To make 100 ml. of buffer at the ionic concentrations given above, pi- 
pette 20 ml. of stock solution into a 100 ml. volumetric flask, add the 
specified volume of O.IOOON HCl as given in the table below, and dilute 
to the mark with distilled water. 

ml. of O.IOOON HCl 
pH 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 

2 73.30 70.35 67.85 65.70 63.85 62.25 60.80 59.55 58.45 57.40 

3 56.50 55.70 54.95 54.30 53.70 53.20 52.65 52.10 51.55 51.02 

4 50.50 49.97 49.45 48.91 48.35 47.80 47.26 46.75 46.22 45.68 

5 45.18 44.60 44.05 43.50 42.94 42.36 41.80 41.23 40.61 40.00 

6 39.42 38.94 38.09 37.45 36.74 36.06 35.36 34.65 33.92 33.25 

7 32.65 31.98 31.45 30.83 30.35 29.87 29.43 29.05 28.68 28.33 

8 28.02 27.69 27.45 27.25 26.90 26.60 26.10 25.63 24.90 24.33 

9 23.75 23.05 22.38 21.72 21.12 20.52 19.94 19.37 18.81 18.35 

10 17.92 17.43 16.97 16.64 16.36 16.15 15.95 15.70 15.40 15.02 

11 14.52 13.93 13.20 12.30 11.23 10.00 8.40 6.60 4.70 2.06 

12 0.40 



•49- 



CHAPTER VIII. 

SALINE AND ARTIFICIAL SEA WATER SOLUTIONS 

SALINES AND SEA- WATERS: GENERAL 

Artificial salt solutions have been prepared which attempt to dupli- 
cate both the osmotic and ionic properties of the internal or external 
environments of given organisms. The aquatic media in which animals live 
are replaced by artificial sea-waters, while the Ringer's and associated 
solutions are substitutes for the tissue fluids (and/or plasma) of the 
various species. In many cases, it suffices to use a solution of a single 
compound which has the same osmotic pressure as the medium, a so-called 
isotonic solution. Such solutions, though osmotically normal, are phys- 
iologically imperfect due to ionic imbalance. 

The chief components of these artificial salines are sodium, potas- 
sium, calcium, and magnesium cations, and the anions chloride sulfate, 
and bicarbonate. They are readily added in the form of six salts mixed 
in the proper proportions, and at the proper total concentration. Reagent 
Grade Merck chemicals and distilled water should be used in all cases. 
Merck's special reagent for biological use sodium chloride should be used 
to avoid toxic impurities sometimes found in other brands. The bicar- 
bonate must not be added until the solution is almost diluted to volume, 
nor before heat sterilization (if necessary) or the calcium will precip- 
itate. 

SALT STANDARDS: 

For convenience in making up the artificial sea-waters and phys- 
iological salt solutions, 8 liter stock bottles of LOOM solutions of 
the major components are kept above the Chemist's work bench. These 
are connected to self-filling burettes for ready delivery of the proper 
volumes of the standard salt solutions. 

Sodium chloride, potassium chloride, sodium bicarbonate, and mag- 
nesium sulfate are not hygroscopic, and their solutions may be prepared 
by weighing out the proper amount of the salt as it comes in the bottle, 
and dissolving to volume. Calcium chloride and magnesium chloride are 
too hygroscopic for this procedure, and therefore approximate amounts 
of these salts are dissolved to volume and the resulting solutions stand- 
ardized and adjusted to LOOM. 

The standardization is done by determining the chloride content, 



■51- 



rather than by the difficult analyses of the respective cations. 10 ml. 
of the unknown is pipetted into a 125 ml. erlenmeyer, diluted to 50 ml., 
and titrated with standard silver nitrate (about 1 molar), using three 
drops of dichlorof luorescein indicator. The titration must be done as 
rapidly as possible to avoid masking of the end-point by the purple color 
of reduced silver. The end-point is taken as the first persistent salmon- 
pink color in the mixture. 

The indicator is prepared by dissolving 0.1 g. of dichlorof luorescein 
and 2.5 ml. of O.IN NaOH in 100 ml. of distilled water, and does not work 
in acid solutions below pH 5.5. The silver nitrate is standardized by 
titration against accurately weighed 1.1-1.2 g. samples of reagent sodium 
chloride. The NaCl is placed in a 125 ml. erlenmeyer, dissolved in 50 ml. 
of distilled water, and titrated by the same method as is used on the un- 
knowns. It will take about 20 ml. of silver nitrate to titrate NaCl sam- 
ples of the recommended amount. 

Weight : Volume Factors for Salt Standards 

1.000 Molar gms./ml. ml. /gram 

NaCl 0.05845 17.11 

KCl 0.07456 13.42 

CaCl2»2H20 0.14703 6.81 

MgCl2*6H20 0.20333 4.92 

MgS04.7H20 0.24649 4.06 

NaHCOg 0.08402 11.91 

Solutions Isotonic with Sea Water Salinity 35 °/oo 

NaCl 0.53M 

KCl 0.53M 

CaClg* 2H2O . 34M 

MgClg* 6H2O 0. 37M 

MgS04« 7H2O 0. 90M 

NaHCOg 0.54M 

NaBr 0. 54M 

Na2SO4+10H2O 0. 44M 

CsCl 0.53M 

RbCl 0.58M 

LiCl 0.60M 

C,2H220n 0.81M 



-52- 



Challenger Report on Sea Water: Analysis of Sea Water at Woods Hole: Cation Analysis of Woods Hole 

gm/l (Page, 1927) ^^/^ Sea Water (Shanklin. 1954) 

NaCl 27.213 Na 8.80 Millimols Per Litre 

MgClj 3.807 K 0.412 Na 534.0 

MgS04 1.658 Ca 0.428 K 18.2 

CaS04 1.260 Mg 1.3004 Mg 56.2 

K2SO4 0.863 CI 18.350 Ca 5.8 

CaCOj 0.123 SO4 2.615 

MgBr2 0.076 PO4 0.002 

HBr 0.06 

For Woods Hole dilute 30.8 volumes to 35 volumes. 



ELEMENTS PRESENT IN SOLUTION IN OCEANIC SEA WATER EXCLUSIVE OF DISSOLVED 

GASES* 
Chlorinity = 19.00 0/00 



Element 


Parts per 


Element 


Parts 


per 




Million 




Mill 


ion 


Chlorine 


18980 


Copper 


0.001 


-0.01 


Sodium 


10561 


Zinc 




0.005 


Magnesium 


1272 


Lead 




0.004 


Sulfur 


884 


Selenium 




0.004 


Calcium 


400 


Cesium 




0.002 


Potassium 


380 


Uranium 




0.0015 


Bromine 


65 


Molybdenum 




0.0005 


Carbon 


28 


Thorium 




0.0005 


Strontium 


13 


Cerium 




0.0004 


Boron 


4.6 


Silver 




0.0003 


Silicon 


0.02-4.0 


Vanadium 




0.0003 


Fluorine 


1.4 


Lanthanum 




0.0003 


Nitrogen 


0.006-0.7 


Yttrium 




0.0003 


Aluminum 


0.5 


Nickel 


- 


0.0001 


Rubidium 


0.2 


Scandium 




0.00004 


Lithium 


0.1 


Mercury 




0.00003 


Phosphorus 


0.001-0.10 


Gold 




0.000006 


Barium 


0.05 


Radium 




0.2-3xl0-^' 


Iodine 


0.05 


Cadmium 




present 


Arsenic 


0.002-0.02 


Cobalt 




present 


Manganese 


0.001-0.01 


Tin 




present 


* H. U. Sverdrup, M. W. Johnson, 


and R. H. Fleming, 


The Oceans , Prentice 


Hall, Inc. 


, New York, 1942. 









Nitrogen in combined forms. 

-53- 



ARTIFICIAL SEA WATERS: 

Sea waters all over the world contain the same proportions of the 
various ions, differing only in the total ionic content and therefore 
in total osmotic pressure. The osmotic pressure is proportional to the 
salinity of the sea water, this being defined as the total grams of solid 
per kilogram of sea water when all the halides have been converted to 
chloride, all the carbonate converted to oxide, and all the organic matter 
completely oxidized. The salinity of Woods Hole sea water is 31 0/00±0.5. 
All the formulae given below have been recalculated to the same salinity, 
and therefore the same osmotic pressure, as Woods Hole sea water. 

A true artificial Woods Hole sea-water, called M.B.L. formula, is 
listed in the table below. This contains all the major ions in amounts 
identical with Woods Hole sea water as it comes from the sea water taps, 
except that the fluorides, bromides and iodides have been replaced by 
chloride, the strontium replaced by calcium, and the borate omitted. In 
the Trace M.B.L. Formula, however, these minor elements are included in 
their proper amounts. These formulae have been tested on sea urchin eggs 
by Ethel Browne Harvey and have been found to be both osmotically and 
physiologically satisfactory. They are the recommended formulae for use 
at the M.B.L. 

The other listed formulas are experimental sea waters which were 
used by the investigators whose names are applied to the solutions. They 
all differ from sea water in all components, but contain approximately 
the same amounts of the various ions as sea water (compare with M.B.L. 
formula). The Allen (Pantin) and Brujewicz formulae correspond most 
closely to true sea water, all the others being low in sulfate and 
equivalently higher in chloride. The Van't Hoff formulae are low in 
calcium and lack the buffer action of bicarbonate, whereas, the Challenger 
sea water contains over double the normal amount of calcium. The two 
calcium-free formulae both replace the calcium with sodium so as to main- 
tain osmotic pressure, but the Shapiro formula is also low in magnesium. 

The listed quantities of the various salts are mixed in a 1 liter 
volumetric flask and diluted to the mark with distilled water. It is 
best to weigh out the sodium chloride, but to burette the LOOM solutions 
for all the other components. Ml. of LOOM refers to milliliters of LOOM 
solution of the salt. Be sure to check the notes below the main table for 
any minor constituents which must be added before diluting to volume. 



54- 



TABLE OF ,\HT1FICIAL SEA WATEHS 

All recalculated to salinity of 31 0/00 and osmotically identical with Woods Hole sea water. 

ALlTiOH NaCl KCl CaCl g' aHgO MgClg-eHjO MgS0^.7H20 NaHOOg 

ml. ml. ml. ml. ml. ml. 

LOOM g/1 LOOM g/1 LOOM g/1 LOOM g/1 LOOM g/1 LOOM g/1 

Allen (same as Pantin)* 423.0 24.72 9.10 0.68 9.50 1.40 23.50 4.78 25.53 6.30 1.15 0.097 

Brujewicz (1) 419.9 24.51 9.00 0.67 9.50 1.40 23.75 4.82 25.38 6.26 2.23 0.187 

Challenger (2) 395.0 23.10 8.88 0.66 21.15 3.12 31.36 6.36 16.78 4.14 xxxx xxxxx 

Horstadius-Bialascewicz (3) 430.5 25.17 8.73 0.65 9.76 1.44 28.36 5.78 18.35 4.53 2.18 0.183 

Horstadus-Roscoff (3) 428-0 25.02 9.80 0.73 9.97 1.47 29.35 5.96 18.07 4.47 2.38 0.200 

M.B.L. Formula (4) 423.0 24.72 9.00 0.67 9.27 1.36 22.94 4.66 25.50 6.29 2.15 0.180 

Moore Calcium-free ** _136._g 25.48 9.68 0.72 xxxxx xxxx 34.13 6.94 16.67 4.11 xxxx xxxxx 

Shapiro Calcium-free ** 442.5 25.88 9.83 0.74 xxxxx xxxx 24.58 5.00 16.80 4.14 xxxx xxxxx 

Trace M.B.L. Formula (5) 423.0 24.72 8.27 0.62 9.27 1.36 22.94 4.66 25.50 6.29 2.15 0.180 

Van't Hoff alpha (6) 430.5 25.17 9.40 0.70 7.22 1.06 34.31 6.96 16.52 4.07 trace 

Van't Hoff beta (6) 432.0 25.23 9.54 0.71 4.36 0.64 33.78 6.86 16.52 4.07 xxxx xxxxx 

Notes and References for Table Above: 

•) No references found except for Formulae and Methods III. 

1) Subow N.N.: Oceanographic Tables, Moscow (1931) • Add NaBr 0.077 g/1 

2) Adapted from the Challenger Report - Add Na2003 1.16 g/1 and MgBr2'6H20 0.11 g/1 

3) Publ. Staz. Zool. Napoli 14 253-429 (1935) 

4) Calculated by J.D. Ostrow from Table of Major Constituents of Sea Water, Lyman & Fleming, 
J. Marine Research 3 134-146 (1940). Tested by Ethel Browne Harvey, M.B.L. 1951. 

5) Calculated by J.D. Ostrow from Table of Major Constituents of Sea Water, Lyman & Fleming, 
J. Mar. Res. _3 134-146 (1940). Add KBr 0.089g. ; NaF O.OOSg.; .SrCl2*6H20 0.037g.; and 
HoBO, 0.024g. per liter of solution. 

6) Original reference for both forms is Van't Hoff, J.H. , Physical Qiemistry in the Service 
of the Sciences, p. 101. Univ. of Oiicago Press (1903); alpha formula from CO. Rogers 
Textbook of Comparative Physiology (1927) N.Y.; beta formula from Osterhout. W.J.V. , 
Bot. Gaz., 42 127 (1906) 

PHYSIOLOGICAL SALINE SOLUTIONS 

In working with isolated tissues, it is important to bathe the 
preparation in balanced saline solutions which duplicate the ionic com- 
position of the tissue fluids and plasma of the donor animal. Such solu- 
tions are known as Ringer's solutions, after the man who first stressed 
the importance of ions in physiological function. The following table 
is a completely revised list of balanced salt solutions, including the 
most recent formulae available. 

The Boyle-Conway Amphibian Ringer and the Krebs Mammalian Ringer are 
recommended by Dr. Szent -Gyoergy i . They are almost identical in every 
respect with the plasma of those two classes, except for the protein com- 
ponents, which are not included in the synthetic media. These will be 
our stock Ringers for amphibians and mammals. The Locke Mammal Ringer 
is the old, standard formula for work on mammalian hearts, whereas the 
Tyrode Mammal Ringer is better for gut muscle. Two Molluscan Ringers 
are included, one for marine animals (determined for mussel) and one for 
fresh water animals (determined for Helix). 



55- 



Four other solutions of a Ringer type are listed. They are given 
in Formulae and Methods III, but original literature could not be located. 
Be sure to check notes below table for other components. 









PHYSIOLOGICAL SALINE SOLUTIONS 








nmGER' S SOLUTIONS 




NaCl 
ml. 

LOOM g/1 


KCl 

ml. 

LOOM g/1 


CaCl2«2H20 

ml. 
LOOM g/1 


MgCl2'6H20 NaHCDj 

ml. ml. 
LOOM g/1 LOOM g/1 


Dextrose 

g/1 


Amphibian (regular) 


(1) 


111.2 


6.50 


1.88 


0.14 


1.08 


0.16 


XXX XX xxxx 


2.38 


0.20 


XXX 


Amphibian (Boyle -Conway- 2) 


72.6 


4.24 


1.99 


0.15 


xxxx 


xxxx 


xxxxx xxxx 


25.01 


2.10 


XXX 


Crayfish (3) 




205.3 


12.00 


5.37 


0.40 


13.55 


1.99 


2.61 0.53 


2.38 


0.20 


XXX 


Oustacean (4) 




525.0 


30.65 


13.27 


0.99 


12.39 


1.82 


24.78 5.04 


to pH 


7.0 


xxx 


Elasmobranch (5) 




280.2 
154.0 


16.38 
9.00 


11.94 
2.68 


0.89 
0.20 


10.00 
1.84 


1.47 
0.27 


xxxx xxxx 


4.52 


0.38 


LOO 


Insect (6) 


xxxx xxxx 


to pH 


7.2 


4.00 


Mammal (Krebs) (7) 




118.4 


6.92 


4.70 


0.35 


2.52 


0.37 


xxxx xxxx 


25.01 


2.10 


xxx 


Mammal (Locke) (8) 




154.0 


9.00 


5.64 


0.42 


2.16 


0.32 


xxxx xxxx 


2.38 


0.20 


2.00 


Mammal (Tyrode) (9) 




136.9 


8.00 


2.68 


0.20 


1.84 


0.27 


1.03 0.21 


11.91 


1.00 


1.00 


Molluscan (Marine) 


(10) 


530.0 


30.97 


10.70 


0.80 


13.00 


1.91 


xxxx xxxx 


xxxx 


xxxx 


xxx 


Molluscan (Fresh-water : 1 1 ) 


98.2 


5.74 


11.94 


0.89 


17.97 


2.64 


27.64 5.62 


24.42 


2.05 


xxx 


Other Solutions* 
























Belar's 




154.0 


9.00 


2.68 


0.20 


1.84 


0.27 


xxxx xxxx 


2.38 


0.20 


4.00 


Clark's (12) 




111.2 


6.50 


1.88 


0.14 


1.08 


0.16 


xxxx xxxx 


1.19 


0.10 


xxx 


Holtfreter's 




59.9 


3.50 


0.67 


0.05 


0.90 


0.10 


xxxx xxxx 


2.38 


0.20 


xxx 


Knowlton's (13) 




220.0 


12.90 


6.97 


0.52 


3.96 


0.58 


S.05 1.02 


xxxx 


xxxx 


xxx 



Notes and References for Table of Physiological Salines 

•) No reference found except Formulae and MethodsIII. 

1) Winton and Bayliss; Human Physiology, p. 393, 2nd Ed. Blakistoh (1935). Add NaH2P04'H20 0.012 g/1 

2) Boyle and Conway; J. Physiol., 100^ 1 (1941). Add MgS04.7H^ 1.22 ml. of IM; Na2S0^' IOH2O 
0.21 g/L Just before use add - NajHPO^ 0.36 g. ; KHgPO^ 0. 07 g. ; Calcium Gluconate 0.40 g. 

3) Harreveld, A. V.; Proc. See. Exper. Biol. & Med., 34, 428 (1936). 

4) Pantin; J. Exper. Biol. , _11^ 11 (1934). 

5) Babkin, B.P. et al. ; Contr. Canad. Biol. & Fish N.S. ,_8^ 209 (1933). Add urea 21.6 g.; 
NaH2P04«H20 0.07 g. 

6) Pringle, J.W.S.; J. Exper. Biol. , 15, 144 (1938). 

7) Krebs, A.; Hoppe-Seyler's Zeitschr., 210, 33 (1932). Add Mg304«7H20 1.18 ml. of IM; 
KH2PO4O.I6 g/1 

8) Locke & Rosenheim; J. Physiol., _36_, 208 (1907). 

9) Gellhorn, E. ; Lehrbuch der Allgem. Physiol, p. 74, Tliieme (Leipzig 1931). Add NaHjPO^'HgO 0.06 g/1. 

10) Singh, I.; J. Physiol. £2^ 62 (1938). Add phosphate buffer at pH 7.2 to strength desired. 

11) Bernard and Bonnet; C.R. Soc. Biol. Paris, 103, 1119 (1930). 

12) No reference. Add NaH2P04'H20 0.012 g/1 

13) No reference. Add urea 20.0 g. Used for perfusing dogfish heart. 



56- 



CHAPTER IX. 
PHOTOGRAPHIC SOLUTIONS 



General Information: 



Stock bottles of all the basic photographic solutions are kept in 
the Chemical Room, from which they may be issued to investigators. Only 
the formulas listed in this manual are kept in stock, all others being of 
a more specialized nature and prepared by the Chemist on order. 

Photo solutions are generally unstable to air and deteriorate on stand- 
ing if left exposed to the atmosphere. They are also unstable to light. For 
these reasons, they are always stored and issued in brown-glass bottles, which 
are initially filled to the top. Orders are drawn from already opened bottles 
before a fresh, completely-filled bottle is opened. As a guide to the inves- 
tigator, a table of the keeping properties of photo solutions is included in 
this manual. 

Preparation of Solutions - General: 

The following list includes all the chemicals necessary for the prep- 
aration of the formulas in this manual. In every case, photo or technical 
grade materials are satisfactory, but distilled water should be used in mak- 
ing all solutions. All formulas are recalculated from those given in the 
Kodak Reference Handbook. 

List of Photo Chemicals 

Elon (Kodak) or Metol (Ansco) Potassium alum 

or Photol (Merck) Chrome alum 

Sodium sulfite, anhydrous Glacial acetic acid 

Hydroquinone Cone, sulfuric acid 

Kodalk (E.K.Co.) Sodium sulfate 

Sodium thiocyanate Sodium thiosulfate. 
Sodium carbonate, monohydrate, (rice cryst.. Hypo) 

NapCOo'HpO Potassium ferricyanide 

Potassium Bromide, KBr Potassium dichromate 
Borax (granular) 
Boric acid crystals 

Preparation of Developers: 

The six stock developers and their uses are listed in the table below: 
D-ll...High contrast for films and plates 



-57- 



D- 19. • .Rapid contrast for films and plates 

DK- 20.. Fine grain for films and plates 

DK- 50 .. General and Professional use on films and plates 

D-72. . .General use for films, plates and papers 

D- 76. . .Maximum speed with normal contrast and 

good shadow detail on films and plates 
Note: ''Microdol'' is DK-20 with preservative added. 

''Dektol'' is D-72 with preservative added. 

Developers are prepared in 8-liter batches. For this purpose, an 
automatic stirrer and a graduated 8-liter jug (with pouring lip) are pro- 
vided for the use of the Chemist. The warm water is heated to about 50 C 
in a 4-liter beaker over a Fischer burner, and then placed in the jug. The 
stirrer is set in motion and the weighed chemicals added in the order listed , 
each constituent being added only after the previous one has dissolved. When 
all the chemicals are in solution, cold water is added to make 8 liters. The 
inaccuracy in judging this from the graduation mark on the side of this jug 
is not crucial. The solution is then poured into the stock bottles with the 
aid of a large funnel provided for this purpose. 

Ingredients D-11 D- 19 DK-20 DK-50 D-72 D-76 

Distilled Water (50**C) 4L 4L 6L 4L 4L 6L 

Elon, Metol, or Photol 8.0 g 17.6 g 40.0 g 20.0 g 24.8 g 16.0 g 

Sodium Sulfite, anhydrous 600 g 768 g 800 g 240 g 360 g 800 g 

Hydroquinone 72.0 g 70. 4 g xxxx 20.0 g 96.0 g 40.0 g 

Kodalk xxxx xxxx 16.0 g 80.0 g xxxx xxxx 

Sodium Thiocyanate xxxx xxxx 8.0 g xxxx xxxx xxxx 

Sodium Carbonate, Monohydrate 234 g 449 g xxxx xxxx 632 g xxxx 

Potassium Bromide 40.0 g 40.0 g 4.0 g 4. g 15.2 g xxxx 

Borax (granular) xxxx xxxx xxxx xxxx xxxx 16.0 g 

Dissolve completely and dilute to 8 liters with cold distilled water. 

Preparation of Acid Fixing Bath with Hardener F-5: 

This is the only acid fix kept in stock since it is the general purpose 
fixing bath, suitable for films, papers, and plates. For reasons of stability, 
it is stored as two solutions which are mixed to order. One is a 30% Hypo 
(Sodium Thiosulfate) solution and the other is the Hardener F-5A. Both are 
kept in bottles on the pressure system, and mixed in a graduated cylinder in 
the ratio 1 part Hardener to 4 parts Hypo. Solutions must be cool before 
mixing. 



•58- 



Preparation of Hardener F-5A: (8 liter batch) 

This is mixed in the same jug with the same stirrer as was used on 
the developers. Ingredients are added in the order listed below: 

1. Distilled water (50°C) 4800 cc. 

2. Sodium sulfite, anhydrous 600 g. 

3. Acetic acid, glacial 513 cc. 

4. Boric acid crystals 300 g. 

5. Potassium alum 600 g. 

6. Cold water to make 8 1. 

Preparation of 30% Hypo: 



If making a 12 liter batch use 3600g of the Hypo(Sodium Thiosulf ate ) , if 
making an 18 liter batch use 5400g of the Hypo. By dissolving the Hypo in sep- 
arate batches the crystals may be made to go into solution more easily and more 
quickly. Therefore, if making a 12 liter batch use two portions of distilled 
water of 6 liters each int.o which are dissolved 1816g (41bs.) and 1784g (3.91bs.) 
respectively. If making an 18 liter batch it is best to dissolve the Hypo in 
three separate 6 liter batches with 1816g (41bs.) of the Hypo in two of the three 
batches and 1784g (3.91bs.) in the third batch. It is easiest and more conve- 
nient to pour the one pound boxes of the Hypo directly into the stirring jug as- 
suming them to be fairly accurate pounds. Any colloidal cloudiness will clear up 
in a day or two while in the carboy. However, the Hypo can be used even though 
it is cloudy. The cloudiness does not affect its effectiveness. Each 6 liter 
portion is poured into the carboy and then the carboy is placed under the pres- 
sure system beside the hardener. 

Preparation of Stop and Hardening Baths: 



1. Stop Bath SB-1: (1.3% Acetic acid) 

For papers and plates. 13 cc. of glacial acetic 
acid in 1 liter of water. 

2. Hardening Bath SB- 3: (3% Chrome alum) 

For use at summer room temperatures with films and 
plates. 30 g. of chrome alum dissolved in 1 liter 
of water. 

3. Stop Bath SB-5: (Non-swelling acid rinse for Photo 

finishing) 

Water. 500 cc. 

Acetic acid, glacial 9 cc. 

Sodium sulfate crystals .. 105 g. 
Water to make 1.0 1 



-59- 



Miscellaneous Formulae: 

Farmer's Reducer R-4A: (Cutting - for clearing shadow areas) 



Stock Solution; 



Dissolve 37.5 g. potassium ferricyanide in enough water to make 500 cc, 
For use, take 30 cc. of stock, 96 cc. of 30% Hypo, and enough water to make 
1 liter. Mix the stock and hypo, then add the water and pour the mixed sol- 
ution at once over the negative to be reduced. When sufficient reduction 
has occurred, remove the negative and wash thoroughly. The mixed reducer 
will keep only briefly. 

General Tray Cleaner TC-1: 

Dissolve 82 g. of potassium dichromate in 910 cc of hot water. Cool, 
and then add slowly with constant cooling and stirring, 87 cc. of cone, 
sulfuric acid. 

Keeping Properties of Solutions: 



The figures given are for no loss in quality on standing without use. 
Developers except D-72: 



1. In tray - 24 hours. 

2. In gallon tank - 1 month. 

3. In stoppered bottle, full 



6 months. 



half-full - 2 months. 



D-72; 



1. Tray - 24 hours. 

2. Gallon tank - 2 weeks. 

3. Stoppered bottle, full - 3 months. 

half-full - 1 month 

Stop-Baths: (SB-1, 3, 5) 

1. Tray - 3 days. 

2. Tank - 1 month 

3. Stoppered bottle - indefinitely 

Acid-Fixing Bath F- 5 : 



1. Tray - 1 week 

2. Gallon tank - 1 month 

3. Stoppered bottle, full - 3 months. 

half-full - 2 weeks 



-60- 



Useful Life of Photo Solutions: 

This table is given in terms of the number of 8 X 10 inch sheets which 
can be processed per gallon of developer without impairment of the quality 
of the solution. 



Solution 

Developer D- 11 
Developer D- 19 
Developer DK-20 
Developer DK-50 
Developer D-72 



8"X 10" sheets/gal. 



Developer D-76 
Acid Fixing Bath F-5 
Stop Bath SB-1 
Hardening Bath SB-3 
Stop Bath SB-5 



Tray 






Dee 


p Tank 






20 








40 






30 








60 






20 








30 






20 








40 






20 (1 


1) 






40 (1 


1) 


Neg 


15 (1 


2) 






30 (1 


2) 


Neg 


30 (1 


1) 


Pr 


ints 








30 (1 


2) 


Prints 








25 (1 


4) 


Prints 








20 








30 






00 








100 






75 








75 






25 








25 






00 








100 







-61- 



^Jjl 



■>.*%, 

'-;:'*• ■* 






«#.