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
-1-
analyses are only limits of tolerance they are of little use in comparing
different brands of chemicals which may contain varying amounts of other
impur it ies .
The purest grades include Me r c k ' s " Reagent Grade", Mai inckrodt ' s
"Analytical Reagent", Eimer and Amend' s "Tested Purity", Baker's
" Analyzed", etc.
Reagent grade and special chemicals are to be issued only when
this grade of purity is requested on the order.
The confusion of different grades of materials is to be guarded
against. For example, do not issue immersion oil for clearing oil just
because both are different kinds of cedar oil. Certain of these confusing
substances are specially marked. In the case of expensive materials issue
no more than the amount marked on the bottle unless permission of the
person in charge is given for a greater amount.
SOLUBILITIES:
If a special solution calls for a large amount of a material consult
the table of solubilities, Chapter 5, or for stains. Chapter 4, to see
if solution is possible. The Merck Index, Chemical Rubber Handbook of
Chemistry and Physics, Lange's Handbook of Chemistry, International
Critical Tables , and the Dictionary of Chemical Solubilities are excel-
lent reference books concerning solubilities of compounds. If in doubt,
consult with the person in charge before attempting the preparation.
This may avoid an error and the wastage of much material. Chloretone,
in particular, cannot be made stronger than 0.6% in water at room
temperature .
ACCURACY AND DECIMALS:
Note that accuracy is relative. An error of 0.1 gram in 500 grams
is only a 0.02% error, while the same error in 1 gram is a 10% error.
Use the proper scales or balance for the particular job to be done. If
there is any question as to the accuracy required in an order, consult
with the investigator placing the order or with the person in charge.
When a decimal is to be placed on the label of any preparation conform
to the following rule: Write the decimal to the number of places known
to be correct, and only one doubtful figure. This same rule can be
applied to figures where the known value does not extend to decimals
by writing one doubtful figure and the rest zeros.
ACCURACY AND ERRORS:
Absolute errors x, -x are deviations from the correct values and
their sign is important for correct statement. They are expressed as
2-
correct to two decimals, or to the nearest million, etc. Absolute errors
are more important in addition and subtraction: e.g., in a column of
figures the absolute errors in tlie third place of a sum or a difference
may be great enough to make the second place unreliable. Relative errors
(x, -x)/x are connected with the number of significant figures and are
usually expressed as percentages. These errors are important in multi-
plication and division. In a product or quotient the number of signif-
icant figures is equal to the number in the weakest factor. Many solu-
tions need not be prepared more carefully than 5% while others must be
made with care to insure sufficient accuracy. If in doubt as to the
precision required consult with the investigator or with the person in
charge. This information and that given above is to be used as a guide
by the staff in the use of the equipment in the Chemical Room.
ALCOHOL DILUTION:
For ordinary histological work special strengths of alcohol may
be prepared by taking the number of milliliters of 95% alcohol equal to
the strength desired in a graduate and adding enough distilled water to
make 95 ml. (Example: to prepare 60% take 60 ml. of 95% alcohol and
add 35 ml. of distilled water making 95 ml. of the strength of the
alcohol used . )
CLEANING METHODS:
Scrubbing with a 2% solution of alconox and hot water followed
by a liberal rinsing with tap water will remove most chemicals. A
small amount of trisodium phosphate applied with the fingertips or with
a small brush will remove pencil markings, most greases, xylene, and
films of paraffin. No abrasives should be used in cleaning volumetric
glass ware. When cleaning solution (sulfuric acid-dichromate ) is used
continued rinsing is necessary. Ten rinsings with water, seven of
tap and three of distilled, are necessary for adequate removal of the
dichromate from the surface of the glass. Since chromic acid is toxic
to living organisms, this is important. For many purposes 1 part con-
centrated nitric acid to 9 parts water is superior to chromic acid as
the nitric acid oxidizes organic material without leaving an adsorbed
residue on the glass. Aqua regia will remove what can be removed by
cleaning solution and will wash off completely with tap water. To clean
staining jars use a little dilute hydrochloric acid. For the few dyes
not removed by this, use a strong solution of sodium hydroxide. Another
useful cleaning fluid is 1-5% trisodium phosphate. Rubber stoppers
may be cleaned by boiling in dilute sodium hydroxide, then rinsing with
-3-
water, followed by boiling in dilute hydrochloric acid and finally
tlioroughly rinsing with water.
GLYCINE or GLYCOCOLL is an amino acid used medicinally and is not to
be confused with the poisonous photographic developer glycin,
(p-hydroxyphenylaminoacetic acid).
A MOLAL SOLUTION (m) contains one gram-molecular weight dissolved in
1000 grams of solvent. For ordinary aqueous solutions 1 ml. of water
is used as 1 gram. For other solutions calculate according to density
at the temperature used.
A MOLAR SOLUTION (M) contains one gram-molecular weight in one liter
of solution. Dissolve the material in less than one liter and make up
to one liter in a volumetric flask.
A NORMAL SOLUTION (acidimetry or oxidimetry) contains one equivalent
of the active reagent in grams in one liter of solution. The equivalent
value of any reagent will depend upon the conditions under which the
reagent is employed. It may or may not be the same as a molar solution.
PERCENTAGE SOLUTIONS: Percent means parts in one hundred parts. These
solutions may be made up according to weight, volume, or any combination
of these. For example, a 3% solution of KCl contains 3 grams of the salt
in 100 grams of solution, or in 97ml. of water. A 3% solution can be
made up in any one of three ways: (a) 3 grams of KCl in a total volume
of solution of 100 ml.; (b) 3 grams in 100 ml. of water; (c) 3 grains in
97 grams of water. The third method will give precisely a 3% solution.
The first two methods do not give a 3% solution, but for concentrations
of 3% or less the error is too small to be of significance. For percent-
ages greater than 3% it is best to prepare the solution on the basis of
weight. For example, a 40% solution of NaOH is made by adding 40 grams
of NaOH to 60 grams of water. Some substances, e.g., alcohol, vary in
strength according to percent by weight or volume. Percentage solutions
(by weight) may be prepared with the solution balance. Place the bottle,
or bottle and funnel, on the pan and balance by means of the weight on
the ungraduated beam. Set the weight on one of the graduated beams and
weigh out the solute, then set for the amount of the solution and add
the solvent until the scale is balanced. The beams are graduated to
facilitate the preparation of percentage solutions but the balance
may be used to advantage for the preparation of other solutions.
The dilution of percentage solutions (aqueous solutions by weight)
can be accomplished easily by taking the number of milliliters
(or multiples thereof) of the stock solution equal to the strength
solution desired and adding enough distilled water to make the total
number of milliliters equal to the strength of the stock solution.
Examples, (a) to prepare 7.1% from 18% stock solution use 7.1 ml. of
the stock plus 10.9 ml. of water which makes a total of 18 ml. (b) to
obtain a 0.02% solution from a 0.4% stock solution use 1 ml. of stock
(50 X 0.02) and 19 ml. of water (50 x 0.38) making 20 ml. (50 x 0.02 +
50 X 0.38) = (50 X 0.4) of the required solution.
In general, remember that in diluting solutions the volume of the
concentrated solution times its concentration is equal to the volume
of the dilute solution times its concentration, or
Vol. X Conc.= Volj,x Cone-
To make a 3/8 M solution from a IM solution, take three parts of the
IM solution and dilute to eight parts. For example, to make 500 ml.
of a 3/8 M solution of NaCl from a IM solution take 3/8 of 500 or
186.5 ml. of IM NaCl and dilute to 500 ml. To make 50 ml. of a
1:10,000 solution requires 1:10,000 of 50 or 0.005 grams of the active
ingredient made to 50 ml. with water.
PROOF is the scale used for measuring the strength of alcohol.
Absolute alcohol is 200 proof; and a mixture containing 50% alcohol
by volume is 100 proof (U.S.A.)
A proof-gallon contains an amount of alcohol equal to that in
a gallon of proof spirit (100 proof). A gallon of proof spirit is
one-half alcohol. Wine gallons multiplied by 1.9 equals proof gallons,
DELIQUESCENT CHEMICALS. Bottle tops of the following chemicals
should be dipped in paraffin.
Acetamide
Acid arsenic
Acid chromic
Ac id citric
Acid monochloracet ic
Acid s i 1 icotungst ic
Acid trichloracetic
Aluminum chloride
Aluminum nitrate
Ammonium acetate
Ammonium fluoride
Ammonium thiocyanate
Barium bromide
Barium chloride
Beryllium chloride
Beryllium nitrate
Beryllium sulfate
Calcium bromide
Calcium chlorate
Calcium chloride,
Calcium chloride,
Calcium nitrate
Calcium oxide
Cobalt sulfate
Ferric chloride
Ferric nitrate
Ferrous chloride
Iodides (most forms)
Lithium bromide
Lithium chloride
Lithium salicylate
Magnesium bromide
Magnesium chloride
Magnesium nitrate
Manganese chloride
anhyd Manganese sulfate
cryst Mercuric nitrate
Potassium acetate
Potassium carbonate
Potassium thiocyanate
Sodium arsenate, cryst
Sodium chlorate
Sodium hypophosphite
Sodium selenate
Sodium sulfide
Sodium sulfite, anhyd.
Sodium thiocyanate
Starch
Zinc chloride
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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.
-8-
CZAPEK' S MEDIUM: (for molds) Sucrose 30 g., sodium nitrate 2 g.,
dibasic potassium phosphate 1 g., magnesium sulfate 0.5 g., potassium
chloride 0.5 g. , ferrous sulfate 0.01 g. , water 1000 ml.
FEHLING'S SOLUTION: Solution I. Cupric sulfate 34.65 g. in 500 ml. water.
Solution II. Potassium hydroxide 125 g., Rochelle salts (sodium potassium
tartrate) 173 g. , water to make 500 ml. Solutions mixed in equal volumes
for use.
FIESER'S FLUID: Water 100 ml., sodium hydrosulfite 16 g., sodium
hydroxide 13.3 g., sodium anthraquinone-B-sulf onate 4 g.
FILM PRESERVATIVE: (acetate base) Camphor 4 g., menthol 4 g., oil
eucalyptus 8 g., glycerine to make 125 ml.
GLYCERINE JELLY: Water 60 ml., gelatin 10 g. , glycerine 70 ml., phenol
(cryst.) 0.25 g. Soak gelatin 30 minutes in the water, dissolve with
gentle heat. Add glycerine and phenol and stir until homogeneous. Store
in wide mouth bottle.
GRAMS IODINE: Water 300 ml., potassium iodide 2 g. , iodine 1 g. ' Mix
KI and I2 in a few drops of water. When dissolved add the remaining
quantity of water.
GREEN FILTER SOLUTION: Water 300 ml., cupric sulfate 35 g. , potassium
dichromate 3.5 g. , cone- sulfuric acid 1 ml.
GROUND GLASS SUBSTITUTE: Ether 240 ml., sandarach 12 g. , mastic 2.6 g.,
benzene 160 ml.
HAINES SOLUTION: Water 170 ml., cupric sulfate 2 g. , potassium hydrox-
ide 6.7 g. , glycerine 15 ml.
HAYEM'S SOLUTION: (for microscopic examination of blood) Water 1000
ml., mercuric chloride 2.5 g. , sodium sulfate (anhyd) 25 g. , sodium
chloride 5 g.
HEAT ABSORBING FLUID: 2% aqueous calcium chloride.
HEAT ABSORBING FLUID: Water 1000 ml., Mohr's salt (ferrous ammonium
sulfate) 200 g. Dissolve and filter. If not clear, add 1.7 ml. cone,
sulfuric acid.
HOLTFRETER'S SOLUTION: Water 1000 ml., sodium chloride 3.50 g. , pot-
assium chloride 0.05 g., calcium chloride (anhyd) 0.10 g., sodium bicar-
bonate 0.20 g. When this is to be sterilized do not add the bicarbonate
until ready to use.
-9-
IODINE, TINCTURE: U.S. P. 70% Alcohol 1000 ml., iodine 70 g. , potassium
iodide 50 g.
IODINE, WATER: Water 1000 ml., iodine 0.2 g.
KEEFE'S SOLUTION: (for preserving green organisms) Alcohol 50%
90 ml., formaldehyde 5 ml., acetic acid (glacial) 2.5 ml., glycerine
2.5 ml., cupric chloride 10 g. , uranium nitrate 1.5 g.
KLOTZ SOLUTION: (for preservation of invertebrate animals) Chloral
hydrate 50 g., Carlsbad salts (see above) 50g. , formalin 100 ml., water
to make 1000 ml.
KNOP'S SOLUTION: Water 1000 ml., calcium nitrate 1 g., potassium
chloride 0.25 g., magnesium sulfate 0.25 g. , monobasic potassium phos-
phate 0.25 g., ferric chloride trace.
KNOP'S SOLUTION: (modification) Water 1000 ml., potassium nitrate
1 g. , calcium sulfate 0.5 g. , magnesium sulfate 0.5 g. , calcium phos-
phate 0.25 g. , ferrous phosphate 0.25 g.
LOCKE'S SOLUTION: (for warm blooded animals ) Water 1000 ml., sod-
ium chloride 9 g., calcium chloride (anhyd) 0.24 g. , potassium chloride
0.42 g. , sodium bicarbonate 0.2 g. , dextrin 2.5 g.
LUGKDL'S IODINE: Water 100 ml., iodine 4 g. , potassium iodide 6g'.
Mix KI and I2 with a few drops of the water. When dissolved, add the
remaining quantity of water.
MAYER'S ALBUMEN FIXATIVE: Separate the white albumen and cut it
with scissors to break up the large masses. Filter through coarse
filter paper and add an equal volume of glycerine. A crystal of thymol
acts as a preservative.
MASSART'S MOUNTING MEDIUM: Water 100 ml., glycerine 16 ml., chloral
hydrate 100 g. , gum arabic 50 g. After fixing in distilled water for
24 hours, then 24 hours in 50% chloral hydrate followed by 24 hours in
100% chloral hydrate and then mounted in the above solution. (Courtesy
Dr. Navez)
MILLON'S REAGENT: (for proteins and nitrogenous compounds) Mercury
10 g., concentrated nitric acid 15 ml. Dissolve the mercury in the
acid, then dilute the solution with 2 volumes of water. Let stand 24
hours and decant. Gives a red color with proteins.
MIQUEL'S SOLUTION: I (culture fluid for diatoms) A. Water 100 ml.
magnesium sulfate 10 g., sodium cliloride 10 g., sodium sulfate 5 g.,
-10-
ammonium nitrate 1 g., potassium nitrate 2 g., sodium nitrate 2 g.,
potassium bromide 0.2 g., potassium iodide 0.1 g. B. * Water 80 ml.,
sodium phosphate 4 g., calcium chloride (anhyd) 4 g., cone, hydrochloric
acid 2 ml., ferric chloride 2 g.
•Make as follows: To the sodium phosphate dissolved in 40 ml. of
water are added, first 2 ml. of HCl , then 2 grams of ferric chloride and
then 4 grams of calcium chloride dissolved in 40 ml. of water, taking
care to shake the mixture. There will be a brown precipitate of ferric
oxide on adding the last solution. It should be separated from the
liquid before using. Forty drops of solution A and 10 - 20 drops of
solution B are added to each 1000 ml. of sea water sterilized by keeping
at 70 C for about 20 minutes.
MIQUEL'S SOLUTION: II (May be added to A and B) Sodium silicate
•9 HgO 5 g. , water 100 ml.
MIQUEL'S SOLUTION: III (Allen's modification) A. Water 100 ml., po-
tassium nitrate 20.2 g. B. Water 80 ml., dibasic sodium phosphate
4 g. , CaCl2»6 H2O 4 g. , ferric chloride (melted) 2 ml., cone. HCl 2 ml.
For use, add 2 ml. of solution A and 1 ml. of solution B to each 1000
ml. of sea water.
MOORE'S SOLUTION: Water 1000 ml., ammonium nitrate 0.5 g. , monobasic
potassium phosphate 0.2 g. , magnesium sulfate 0.2 g. , calcium chloride
(anhyd) 0.1 g. , ferric sulfate trace.
NAEGELI'S SOLUTION: (culture medium for fungi) Water 1000 ml., di-
basic potassium phosphate 1 g. , magnesium sulfate 0.2 g., calcium
chloride (anhyd) 0.1 g., ammonium tartrate 10 g.
NESSLER'S REAGENT: (Block and Benedict from Hawk and Bergein)
Mercuric iodide 100 g., potassium iodide 70 g. , sodium hydroxide 100 g.
Place 100 g. mercuric iodide and 70 g. of potassium iodide in a liter
volumetric flask and add about 400 ml. of water. Rotate until solution
is complete. Now dissolve 100 g. of sodium hydroxide in about 500 ml.
of water, cool thoroughly and add with constant shaking to the mixture
in the flask, and make up with water to the liter mark. If a precip-
itate forms, decant the supernatant liquid and use.
PASTEUR'S SOLUTION: Potassium phosphate 2 g., calcium phosphate 0.2 g.,
magnesium sulfate 0.2 g., ammonium tartrate 10 g., cane sugar (sucrose)
150 g., water to make 1 liter.
PHENOL: To liquify add 10 ml. of water to each 450 g. melted phenol.
Melt by immersing bottle in hot water.
-11-
PLATING SOLUTION FOR ELECTRODES: Platinum chloride 1 - 3%, lead ace-
tate 0.02%. Use 4 volts for about 10 minutes.
PYROGALLOL FLUID FOR ABSORBING OXYGEN: Water 500 ml., potassium
hydroxide 220 g., pyrogallic acid 15 g.
POISON IVY PREVENTIVES: I. Saturated aqueous sodium thiosulfate.
II. Ferric chloride 5 g. , glycerine 50 ml., water 50 ml.
III. Ferric chloride 5 g., glycerine 5 ml., 50% alcohol 100 ml.
RAFFEL'S FLUID: Water 1000 ml., potassium nitrate 0.5 g. , dibasic
potassium phosphate 0.06 g., magnesium sulfate 0.02 g., ferric chlo-
ride 0.001 g.
RINGERS SOLUTIONS: Cf. table in Chapter VIII
SALINE: (normal physiological) Water 1000 ml., sodium chloride
7 to 9 grams. For cold blooded animals, use 7 g. For warm blooded
animals use 9 g.
SZOMBATHY'S FLUID: Gelatin 1 g. , water 100 ml., sodium silicate
0.02 g. , glycerine 15 ml. Dissolve the gelatin at 30 C. Cool and
filter through cloth.
TOISSON'S MIXTURE: (a diluting fluid for blood) Water 160 ml.,
sodium sulfate 8 g., sodium chloride 1 g., glycerine 30 ml, j methyl
violet 0.025 g.
TYRODE'S SOLUTION: (for gut muscle, no advantage for heart muscle)
Water 1000 ml., sodium chloride 8 g. , potassium chloride 0.2 g.,
calcium chloride (anhyd) 0.2 g., magnesium chloride 0.1 g., monobasic
sodium phosphate 0.05 g., sodium bicarbonate 1 g. , glucose 1 g.
ULTRA-VIOLET LIGHT is removed by saturated aqueous sodium nitrate
or 10% CuS04»5 H2O.
VAN DER CRONE'S SOLUTION: (green organisms) Water 1 liter, pot-
assium nitrate 1.0 g., magnesium sulfate and calcium sulfate each
0.5 g., calcium phosphate 0.25 g. , ferrous phosphate 0.25 g. Make
slightly acid or neutral with phosphoric acid.
VAN'T HOFF'S SOLUTION: (artificial sea water) Sodium chloride 19.0
g. , magnesium sulfate 1.5 g., magnesium chloride 2.4 g., potassium
chloride 0.53 g. , calcium chloride (anhyd) 0.37 g., anhydrous salts
dissolved and made up to 1000 ml. with glass distilled water. Also
Cf. table in Chapter VIII.
-12-
CHAPTER III.
KILLING AND FIXING FLUIDS
COPPER ACETATE FORMALIN: Saturated cupric acetate in 40% formalde-
hyde. Dilute to about 4% for preservation of green algae.
CUPRIC-PARANITROPHENOL FLUID: (Petrunkevitch) 60% alcohol 100 ml . ,
nitric acid 3 ml., ether 5 ml., cupric nitrate (3 HpO) 2 g. , para-
nitrophenol cryst. 5 g. One part formaldehyde may be added to 4-7
parts of the fluid just before use.
CUPRIC PHENOL FLUID: (Petrunkevitch) _A^: distilled water 100 ml.,
nitric acid 12 ml., cupric nitrate (3 HgO) 8 g. _Bj 80% alcohol 100
ml., phenol cryst. 4 g. , ether 6 ml. Use 1 part of j\^ to 3 of B.
Will not keep after mixing.
FAA: (General Biological Supply House) 50% alcohol 100 ml., 40%
formaldehyde 6.5 ml., glacial acetic acid 2.5 ml.
HOLLANDE'S FLUID: Formalin sat. with picric acid 12 ml., absolute
alcohol 54 ml., benzene 3 ml., and nitric acid 1 ml.
LANE'S SOLUTION: Potassium bichromate 2.5 g., mercuric chloride
5.0 g. , water 100 ml.
NAVASCHIN'S FLUID: 10% chromic acid, 1.5 ml., glacial acetic acid
1 ml., formaldehyde (40%) 0.83 ml., water 32.67 ml.
PRESERVING FLUID FOR GREEN ALGAE: Potassium chrom alum 10 g.j form-
aldehyde 5 ml., water 500 ml.
STOCKARD'S SOLUTION: Water 85 ml., formalin 5 ml., glacial acetic
acid 4 ml., glycerine 6 ml.
SUSA'S FLUID: Water 80 ml., mercuric chloride 4.5 g., sodium chloride
0.5 g., trichloracetic acid 2.0 g., formalin 20 ml., glacial acetic
acid 4 ml.
WORCESTER'S FLUID: 10% formalin saturated with mercuric chloride,
90 ml., glacial acetic acid, 10 ml.
OSMIUM AND PLATINUM CONTAINING FLUIDS:
Osmium and platinum fixatives are costly and often do not keep
13-
well. Few cytologists use the same formulae, each usually wanting
his favorite formula, hence it is best to keep on hand certain stock
solutions, among which are small amounts of osmic acid and platinic
chloride. Below are listed certain fixatives containing one or both
of these reagents, also a list of stock solutions. The makeup of the
fixatives from the stock solutions is given in parts by volume, and the
amount desired by an investigator can be made up to the nearest mul-
tiple of the total parts indicated.
Acetic acid, glacial
Chromic acid, 1%
Chromic acid, 1% in
1% NaCl
Formic acid
Mercuric chloride, sat
soln. in hot water
STOCK SOLUTIONS
Mercuric chloride, 0.5%
in 1% chromic acid
Osmic acid, 2%
Picric acid, sat. aq.soln.
Platinic chloride, 10%
Potassium dichromate, 10%
FIXATIVES:
Some of the fixatives listed here keep well and may be kept for
a long time. Those which deteriorate are noted. All of these form-
ulae are from Lee's Vade Mecum, unless otherwise stated. In making
osmic acid wash off the paper covering of the glass ampoule; rinse
in distilled water, and file a notch around the tube. Drop the am-
poule into a clean, glass stoppered bottle of a capacity greater
than the amount of osmic desired. The tube of osmic crystals may
now be broken open with a heavy glass rod. As many tubes as wanted
may be crushed inside the glass bottle but not over 200-300 c.c. of
2% should be kept in solution.
All osmic acid and fixatives containing it should be kept in
dark bottles with well fitted glass stoppers. Osmic acid reduces
slowly in the light and at high temperatures; when it is issued it
should be in a brown bottle or the bottle should be covered with black
paper .
When issuing fixatives the label should indicate definitely the
formula used, since there are 4 Flemming's and 3 Von Rath's solutions.
14-
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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 0 (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 0
(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
0
0
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 0
(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 0
(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.
0
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
0
2.732
Mgl2+8H20/MgCl2+6H2
0
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.
0
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 0 . IM 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 0 . 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 0 . 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. 0 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
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