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

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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

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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

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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

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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

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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.

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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.

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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.,

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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.

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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.

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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-

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-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

■>.*%,

'-;:'*• ■*

«#.