THE BRITISH HO~I(EOPATHIC JOURI~AL 7

BIOCHEMICAL AND BIOLOGICAL EVIDENCE OF THE ACTIVITY OF

HIGH POTENCIES*

W. E. BoYD, M.A., M.D., M.Brit.I.R.E.

SUMMAa~Y
1. A m e t h o d is described for investigating the possible action of microdoses of mercuric
chloride on the hydrolysis of soluble s t a r c h w i t h m a l t diastase.
2. The microdoses of the mercuric chloride used in the latest crucial series carried o u t in
1946, 1948, a n d 1952, were w h a t are t e r m e d " h i g h potencies" m a d e in accordance w i t h
the p h a r m a c e u t i c a l m e t h o d of p r e p a r a t i o n of drugs ordinarily used in the practice of
homceotherapy.
3. These microdoses were p r e p a r e d b y separate stages of dilution, the solution at each stage
being subjected to mechanical shock. The solutions were, theoretically, "dilutions" of the
order of 1 in l0 -~1 and on p r e s e n t physical t h e o r y would n o t contain a n y molecules of the
original mercuric chloride.
4. The difference in rate of hydrolysis b e t w e e n flasks containing starch, diastase, a n d dis-
tilled w a t e r (controls) a n d flasks containing starch, diastase and mierodoses of mercuric
chloride (tests) were c o m p a r e d colorimetrieally b y the Spekker a b s o r p t i o m e t e r , a n d the
frequencies of the differences statistically analysed, as the results obtained showed bio-
logical scatter. More t h a n 500 such comparisons were carried out. The differences of m e a n s
were examined b y the Fisher " t " test, the variances tested and Cochrane and Cox's
test applied where indicated. All the series gave a highly significant difference in the
rate of hydrolysis b e t w e e n controls a n d tests, the microdoses s t i m u l a t i n g the process.
Statistically the significance is s h o w n b y the fact t h a t a probability of <0. 001 w a s
obtained independently in each of the three years 1946, 1948 a n d 1952. The control results
gave an a p p r o x i m a t e l y n o r m a l distribution.
5. The distribution, control m e t h o d s , a n d accessory control procedures were considered to
exclude, as a cause of the effects, adsorption of the original d r u g a n d the presence of
e x t r a n e o u s c o n t a m i n a n t s b y chance solely in test flasks. The only difference b e t w e e n
control a n d microdose flasks was the addition of microdose, t h e distilled w a t e r being
c o m m o n to b o t h controls a n d tests.
6. I t w a s concluded t h a t a factor, unidentified, derived from the mercuric chloride used, was
present in solutions p r e p a r e d b y serial dilution w i t h mechanical shock which could affect
the distilled w a t e r diluent, t h a t this change was transferable to s u b s e q u e n t "ultra-
molecular" stages of " d i l u t i o n " , a n d t h a t this factor was the source of the activity in t h e
microdose solutions producing the acceleration of the rate of hydrolysis.
7. I n an a d d e n d u m there is described recent biological w o r k which is also providing evidence
of t h e presence of an active selective factor in " h i g h p o t e n c i e s " derived f r o m S t r o p h a n t h u s
s a r m e n t o s u s b y the same m e t h o d s of dilution w i t h mechanical shock.
The primary purpose of a series of investigations carried out under m y
direction over the last fifteen years has been to determine whether microdoses
of the order of "potencies" as used in the practice of homceotherapy could have
any effect on an enzymatic process. The estimate of the rate of hydrolysis of
starch with diastase was adopted as a possible means of detecting any such
effect.
The drug used throughout has been mercuric chloride, which for this
particular purpose was first used by Persson (1932, 1933) working in the Bio-
chemical Institute of Leningrad under Professor Ginsberg. His technique,
obtained from his papers and from personal communications, gave results which
on occasion were sufficiently suggestive to justify plans for a serious investiga-
tion under more carefully controlled conditions.
Our basic technique was first developed in the Trust Laboratories by
Dr. Pettigrew and Dr. Herd, both Barbour l~esearch Scholars in Biochemistry
at Glasgow University. Later experimental work led to improvement in tech-
nique and controls following a very helpful personal communication in 1939
from Professor Hopkins. A standard range of experimental error was arrived at
and no crucial experiments were undertaken until a large number of prior control
tests were found to be close to these limits. This point is emphasized because
the rate of hydrolysis of starch with diastase will v a r y considerably unless
carefully controlled conditions are present.
* P r e s e n t e d in a b b r e v i a t e d form on March 16th, 1954, to the Scottish B r a n c h of the
F a c u l t y on b e h a l f of the B o y d Medical Research T r u s t I n s t i t u t e , Glasgow.
8 THE BRITISH HOM(EOPATItIC JOURNAL

The amount of detail given in this paper and its appendices has been found
necessary both to meet requests from interested research workers and in order
to supply sufficient information regarding the control methods to make possible
adequate assessment. This is especially desirable because the results are difficult
to explain theoretically.
The drug was made from a good pharmacopoeial brand of mercuric chloride,
as the aim of the research was to study the action, if any, of a drug as it might
ordinarily be used. To use a highly purified chemical was, therefore, not con-
sidered advisable for crucial tests, but the same type of result was also obtained
with an Analar preparation. Whether or not the additional concomitants in the
original drug entered into the effects obtained, the methods of control, including
spectrographic analysis, indicated that the results were related to the nature of
the added microdose, derived from the mercuric chloride.
In the experiments to be described the term microdose is used to designate
a so-called "high potency" of mercuric chloride prepared in general conformity
with the homoeopathic pharmacopoeial method (Leeser, 1938). This method of
preparation, involving series dilution in separate bottles with succussion (mech-
anical shock), results in a solution which does not, from the standpoint of present
physical theory, contain a molecule of the original mercuric chloride prepara-
tion, provided no adsorption from prior use is present. Calculation shows
that after a dilution of approximately l0 -22 none of the ordinary primary
substances remains. The microdoses actually used termed $27 to $31 were
theoretically of the order of 10-61 to 10-71 (Appendix F). This apparent contra-
diction in terms will be discussed later.
I n estimating enzyme activity there are, broadly speaking, two main
procedures available. In the one the amount of chemical change over as short
an interval as possible from the onset of the reaction is measured thus ensuring
nearest approximation to constant conditions. I n this way the activity of the
enzyme is arrived at. I n the other, the length of time required to produce a given
amount of chemical change is measured. The conditions of the experiment are
kept as constant as possible, the enzyme requiring to be stable under the
selected conditions.
In the experiments to be described, however, the primary aim was not to
measure the activity of a single enzyme, but to find whether a microdose might
affect the rate of chemical change of an enzyme process, in this case, the hydro-
lysis of starch with diastase. I t was necessary to have a controlled process
sufficiently constant to make possible an accurate comparison of the rate of
chemical change in two flasks the one containing, as an addition, microdose,
the other, distilled water.
The procedure preferred was to use a fixed time of incubation and to obtain
a colorimetrie comparison of the stage reached by the chemical change in each
of the flasks at the end of the selected time. I t was found possible, by using a
less active enzyme mixture, stable under the conditions laid down, to obtain
such comparisons with a longer incubation time than usual. This longer time, as
will be seen, ensured very accurate timing and careful measurement of small
scattered differences. I t also enabled eight pairs of experiments to be carried
through in one session, under identical conditions, which proved to be the best
method to provide sufficient results for statistical assessment.
The colorimetrie assessment of the stage of chemical change reached
in the given incubation time, and thus of the over-all rate of hydrolysis, was
obtained by measurement, with the Spekker absorptiometer, of the absorption
of green light by the test or control solutions after the addition of Lugol iodine.
A malt diastase was used with Lintner's soluble potato starch as substrate.
Although malt diastase is a relatively crude substance containing, as far as is
known, at least 5 enzymes, it was found that it could be adequately controlled
and gave the desired slower rate of hydrolysis, while having the necessary
thermo-stability.
 EVIDENCE OF THE ACTIVITY OF IIIGH POTENCIES 9

Experiments with a bacterial s-amylase showed t h a t if one used a dilution

sufficient to give the longer incubation time required, the thermo-lability was
too great. Various proportionate mixtures of this s-amylase and F-amylase, both
obtained from U.S.A., did not indicate t h a t any advantage was to be gained.
Bacterial amylases have for some time been extracted from b. subtilis culture on
a suitable medium for industrial purposes, while Carlo and l~edfern (1947)
isolated e-amylase in a purified state from an industrial product. I t has been
obtained in crystalline form by Meyer, Fuld and Bernfeld (1947). As b. subtilis
was cultured from the malt diastase solutions used, the possibility of any hydro-
lytic action being due to its presence was investigated. Under the experimental
conditions of the technique described no effect was found. When necessary the
b. subtilis could be removed by sintered glass filtration. (See Appendix D1 on
bacterial investigations.)
The amylolytic enzymes which take part in the total digestion of starch are
s-amylase, F-amylase, Z-enzyme, and R-enzyme, recently found in potato, with,
in this case where malt diastase was being used, maltase. The part these enzymes
play is discussed later. I t might be argued t h a t the use of crude diastase might
result in fractions of the diastase powder having unequal activity. As will be
seen the plan of the experiment allowed for small differences from day to d a y
within set limits, but for each day the one solution was made up and was com-
mon to all flasks, both control and microdose. The greatest difficulty was to
obtain malt diastase without artificial loading which was sufficiently thermo-
stable for the period of each experimental session. A series of batches had to be
tested using sampling methods until a suitable one was obtained.
The technique required to deal adequately with known factors which
might profoundly modify such a sensitive reaction, and also to take care of any
unknown factors by adequate control methods. The known factors requiring
careful consideration were the constancy of the pH, the time from preparation
of the diastase solution, the time of incubation, the temperature of the bath, the
temperature of the solutions in the flasks, the accuracy of the method of the
estimation of the colour reaction obtained, and the effect of COs concentration
on the solution in the flasks.
Previously in these laboratories, microdoses of mercuric chloride of molar
concentration of the order 10-6, with the quantities of starch and diastase used,
were, as is well-known, found to inhibit the reaction on all occasions. On the
other hand in concentrations of the order of from 10 -9 to 10 -14 molar, a much
more scattered effect was obtained, but statistically in a series of experiments
some stages of dilution, prepared by serial dilution with mechanical shock, were
found to give results suggesting stimulation of the rate of the starch-diastase
reaction (Boyd, 1941, 1942, 1946). In one of these series (1946), evidence was
obtained statistically (P = 0"001) t h a t the procedure of mechanical shock with
dilution had relationship to a stimulative effect obtained with microdoses of the
stage 10 -9 molar, as tests with a comparable set of dilutions prepared without
mechanical shock showed the usual fade out of inhibition at l0 - s and no stage
giving stimulation. This supported the use of this procedure in the preparation
of "high potency" microdoses, apart from the necessity to follow the pharma-
ceutical method under examination.
During one of the series, carried through by Dr. Pettigrew in 1940, a few
tentative sets of experiments were included, somewhat sceptically, using micro-
doses of the order of "high p o t e n c y " solutions. To our surprise one set of
these solutions made in twenty-eight stages, now termed $28 (nominally 10 -64
over a series of twenty-seven pairs of test flasks showed stimulative effects
with a statistical significance (P ~ 0"04) by the t-test. The matter was left in
abeyance for further investigation at a later date when increased experience and
improved equipment might make it possible to retest for a "high potency"
effect with more detailed controls.
A series of tests using the nominal range $26 and $30 were run in 1946. The
 l0 TItE BRITISH HOM(EOPATHI(3 JOURNAL

technician carrying out these runs had increasing experience of the evolution
of the technique over a prior period of four years and was a meticulous and care-
ful worker. The results were statistically highly significant for stimulation.
I n 1948, after a series of further experiments over two years to test the tech-
nique still more carefully, and to introduce semi-automatic suction pipetting,
the same technician was again put on to a new series of $27. Again the result was
statistically highly significant.
I t was felt, however, t h a t until these results could be repeated b y another
independent technician, trained separately in the technique, publication would
not be justified. Plans for further controls were made to include even greater
timing accuracy extending to every part of the technique, including the time
of day when solutions were made and heated before mixing, and additional
checks for bacterial contamination, COs concentration etc. Attempts were made
to train several technicians without success, none of these maintaining the
necessary meticulous care and sense of rigid attention to detail over long
periods of time necessary to attain sufficiently constant control results within
the permitted experimental error. I t is to be understood t h a t "training" does
not refer merely to the use of the absorptiometer and of suction pipetting,
but to continued practice in timing and in the general technique including
the details of cleaning, sterilization, and handling of microdose solutions found
necessary to prevent the appearance of aberrant inhibition in odd flasks at
scattered intervals which might have seriously complicated the assessment of
crucial tests.
I n 1950, however, a new technician was found, whose previous training
had been along lines requiring the same qualities of precision and conscientious
attention to detail as were required, and she was finally trained over about
eighteen months. While not trained for as long as the earlier skilled technician,
her control results over some months fell satisfactorily within the range of
experimental error, and it was decided to proceed to crucial tests. Delay was
experienced due to inability to obtain a fresh supply of a sufficiently thermo-
stable diastase. I n 1952 a new batch of malt diastase was obtained which was
stable and the conditions were felt to be sufficiently satisfactory for crucial tests
to be attempted.
The technique and methods described are those used in the 1952 series
of tests, and are recorded as the most accurate that could be devised for this
particular method. Later in 1952 a physical chemist joined the staff for addi-
tional investigations bearing on the technique and the possible nature of the
microdoses. The 1946 and 1948 results are included in the statistical section,
while any distinctive differences in the methods then used are described in
Appendix E.
GENERAL 1)RINCIPLES
The actual tests consisted simply of comparing, after a given time of
incubation, members of pairs of flasks, the first member containing starch,
diastase, and distilled water, the second containing starch, diastase, and
microdose prepared in the same distilled water. The comparison depended
on the Spekker absorptiometer value obtained with the coloured solution
after the addition of Lugol iodine to each flask. This value, with OG1 green
filters, is designated in these experiments absorptiometer green value, (A.G.V.).
The "test results" were in terms of the differences in A.G.V. between the first
control flask and the second test flask of each separate pair, with the Spekker
set at 0-30 for standard with the first flask. I n addition pairs of control blanks
with starch, diastase and distilled water in both flasks were similarly
measured and the difference values between the first and second flasks of each
pair of blanks provided the "control results" for statistical analysis. These
results thus gave a measure of any small control differences due to experimental
bias in the measurement of second flasks.
 EVIDENCE OF TIlE ACTIVITY OF I-IIGtI POTENCIES 11

In addition, the pairs of flasks were also compared with a distilled water
standard with the absorptiometer set at 1. This provided another set of differ-
ences for comparison and gave the range of variation of the first control flasks of
all the pairs, and also provided a check t h a t the stage of hydrolysis was within
the limited region most suitable for the use of the green filters.
For supplementary information, b y using a sampling procedure with
suitable quantities of the reagents in one large flask, serial absorptiometer
values relative to time could be obtained. I n this way hydrolysis curves with
the green filter could be compiled for various purposes of control, and also to
assess the activity of any batch of diastase and the concentration of enzyme
and substrate required to give the best range of A.G.V. for a given time of
incubation.
I n order to appreciate the nature of the reaction and the interpretation of
results, the hydrolysis of soluble starch with malt diastase, the starch-iodine
complex, and possible factors involved in stimulation require to be considered.
The constitution of the starch substrate and content of the malt diastase
made up of enzymes and accessory substances make the interaction of the two,
leading to hydrolysis, a complex process. A summary of recent knowledge and
theory about them is given in Appendix A and will help those unfamiliar with
the process to picture the nature of the experiments. For the immediate purpose
of the methods to be described it is only necessary to appreciate that the action
of the diastase on the starch leads to a breakdown of the amylose and amylo-
pectin in it. The slightly colloidal starch solution first loses its viscosity, then
there is a change in the iodine reaction colour from blue through purple to red,
then the red colour fades till only the iodine colour remains. Absorption peaks
are at approximately 580 m~, 560 m~ and 520 m~ respectively at the three
colour stages mentioned (Swanson, 1948). Amylose forms a stable blue complex
with iodine, while amylopectin forms a less stable red complex. The structure
of the blue complex is a helical amylose chain of glucosidic units within which
the iodine molecnles are arranged parallel to the helix axis (l~undle and French
1943). Degradation by heating, acids, or enzymes progressively breaks down
amylopectin to produce smaller fragments--only the largest of which still give a
red colour with iodine. The hydrolytic products of starch form highly-coloured
addition products with iodine, the achromic point coming at about six glucose
units, i.e. dextrins of six or less glucose units show no intensification of the
colour of an iodine solution. This is attributed to the inability of the dextrins to
form a complete turn in the helix (Hanes, 1937). The blue colour is given by
chains over 33 units while a red colour is produced by chains of 7-13 units.
Intermediate colours are given b y chain lengths of 13-33 units (Swanson 1948).
The degree of colour change produced in this way on addition of Lugol iodine is
differentiated by the Spekker absorptiometer more clearly if the absorption
measurement is carried out with a suitable spectral band instead of with white
light. The OG1 green filters were found to be the most suitable.
I n practice it was also found t h a t the absorption of the green light in the
Spekker decreased with a resulting increase in the A.G.V., due to the change of
density of the iodine colour reaction as hydrolysis increased. Where such curves
are shown with Spekker readings on the abscissae measured against distilled
water at 1"0, the absorption = 1 -- x where x ~ Spekker reading. While no
exact quantitative relationship between light absorbed with this filter and the
degree of hydrolysis can be deduced with its use, it is possible to obtain a curve
of rate of hydrolysis in terms of the A.G.V. This form of curve is obtainable from
day to day, subject to such factors as might modify the overall rate on different
days. Similar curves can be obtained between two independent runs of sample
tests measured accurately, providing common original solutions are used. I t is
necessary to explain t h a t such curves are never identical and are not sufficiently
constant to provide more than general information. Figs. 1 and 2 give examples
of such double sampling flask runs, using a Merck diastase.
]2 THE BRITISH HOM(EOFATHIC JOURNAL

~2

.8--

~.6 1

0 I
so
I
ss
I
60
I
6s
I
7o
I
7s
I
80
45
"TIME IN MINUTES

FIG. 1
Two n o r m a l s a m p l i n g tests r u n simultaneously. Soluble s t a r c h 5 g. in 500 ml. distilled water.
Merck diastase 0. 045 g. in 100 ml. distilled water, filtered W h a t m a n 40, k e p t cold while
being filtered. The t w o curves are v e r y similar. F i r s t t e s t $. Second test x. These were tests
to assess quantities for a s i x t y m i n u t e incubation period for paired flask tests. E a c h s a m p l i n g
flask contained 240 ml. starch, 48 ml. diastase, a n d 48 ml. distilled water.

"8w

<-6~

'4~
in

O I I I I I I I
4S 50 55 60 b5 70 75 80

TIME IN M I N U T E S

FIG. 2
Two n o r m a l s a m p l i n g tests r u n on separate days. Soluble s t a r c h 5 g. in 500 ml. distilled
water. Merck diastase 0.045 g. in 100 ml. distilled water, filtered W h a t m a n 40, k e p t cold
while being filtered. The t w o curves are closely similar. These were tests to assess quantities
for a s i x t y m i n u t e i n c u b a t i o n time for paired flask tests. E a c h s a m p l i n g flask contained
240 ml. starch, 48 ml. diastase, a n d 48 ml. distilled water.
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 13

The transmission curve for the particular filter used has a peak giving
41-2 per cent. transmission in the region 520 m~ to 540 m~. When this peak
was compared with spectrophotometric measurements at stages in an experi-
ment where Spekker readings were 0" 64 and 0.77 respectively against distilled
water at 1.0, one solution, giving a Spekker reading with this filter of 0" 64, gave
a m a x i m u m absorption of 47 per cent. at approximately 520 m~, while another
solution with a Spekker reading of 0" 77 gave a m a x i m u m absorption of 36 per
cent. also at approximately 520 m~. This filter should give theoretically within
this range the widest differentiation.
I n practice as against others of the H455 set it gave the widest variation in
Spekker reading for a given difference in optical density between two samples
near this range. I f there is an increase or diminution of the rate of hydrolysis
in a test flask when compared with the control flask of the pair, the difference will
show as a greater or lesser Spekker reading respectively with reference to the
green light. The reading, as pointed out above, is comparative. Thus comparative
readings between pairs of control flasks can also provide a measure of experi-
mental error, and provide control differences for statistical assessment.
The term stimulation is used where an increase of rate of hydrolysis is
found in test flasks as against control and refers to the reaction as a whole.
The relative concentration of an enzyme and its substrate plays its part
in varying the rate of reaction. Activation of the substrate is caused by adsorp-
tion or chemical union of enzyme and substrate. This combination is thought to
take place at certain specific points on the surface of the molecules. At such
points the enzyme and substrate are said to "fit", and if this fit is even slightly
imperfect the substrate molecules m a y be under strain, altering slightly the
stereo-chemical pattern, thus making the molecule more chemically reactive.
I n this way hydrolysis m a y occur more readily. The activation of the substrate
does not necessarily result in a chemical modification of substrate such as
hydrolysis, as other substances and processes enter in. These necessary sub-
stances m a y be part of the activating system or part of the reaction system
subsequent to the activation of the substrate. There m a y be activators of
the enzyme itself, which m a y enter in by removal of material t h a t inhibits b y
blocking the reactive centres in the enzyme. Traces of heavy metals m a y play
a part in this activation or inhibition, b y effects depending on polarity producing
alteration in the stereo-chemical pattern of specific centres. I n hydrolysis,
water molecules are involved in the reaction subsequent to the activation by the
enzyme (Baldwin, 1952). I t is thus evident t h a t stimulation of a hydrolytic
process with increased rate might be due to several combinations of factors,
or to some single factor, including the part played by the trace elements such as
sodium, calcium or magnesium, which are present even in carefully distilled
water.
I n the experiments, described, however, these latter elements were present
in the starch, diastase, and to a lesser extent, in the distilled water and were,
therefore, common to all solutions whether control or test.
TH and the Use of Buffers
Normally solutions in such experiments are buffered. I n the crucial experi-
ments, after careful consideration and experiment, it was decided to omit any
buffer. I n view of the fact t h a t we were testing for possible action of either
chance trace quantities of mercuric chloride, or for an unknown "potency
effect" derived from mercuric chloride, it was essential to avoid, if possible,
the introduction of any additional chemical substances which might contain
inhibitory contaminants.
The method required suitable quantities of diastase and starch to give a
rate of hydrolysis to produce after ninety minutes a control A.G.V. within a
moderate range of difference on the Spekker in the region of 0" 60 to 0- 80 against
distilled water at 1.0. This provided sufficient time to examine ~ixteen flasks at
14 THE BRITISH HOM(EOPATHIC JOURNAL

each m a i n test while l i m i t i n g as far as possible the time of t h e diastase in t h e

i n c u b a t i n g b a t h at 37 "5 ~ including the preheating period before addition t o
starch.
The use of a sodium a c e t a t e - - s o d i u m veronal buffer a d j u s t e d b y a d d i t i o n
of 0" 1 N H2S04 definitely slowed this rate for the same q u a n t i t i e s of starch a n d
diastase as used w i t h o u t the buffer. This was clearly i n t r o d u c i n g i n h i b i t o r y
action, as the p H a t which the actual unbuffered experiments were carried o u t
was of the same order as t h a t used with the veronal buffer a d j u s t e d to suit.
As the effect looked for was one of slight s t i m u l a t i o n it was likely to be m a s k e d
b y the i n h i b i t o r y action of the ingredients or c o n t a m i n a n t s i n the buffer. I t was,
therefore, desirable to use the diastase w i t h o u t buffer, provided there was f o u n d
evidence t h a t the solutions were r e m a i n i n g a t each m a i n test a t a sufficiently
c o n s t a n t p H level. E x p e r i m e n t a l findings w i t h o u t buffer provided the necessary
evidence t h a t this was the case.
I t was f o u n d t h a t the A.G.V. variations o b t a i n e d i n 16 flask tests both of
controls a n d test flasks a n d in samples from big flask r u n s at n i n e t y m i n u t e s i n c u b a -
tion, were n o t relative to a n y small differences o f p H , while a 16 flask test r u n in
pairs as normally, except for two m i n u t e s between each m e m b e r of each pair,
showed no p H difference greater t h a n 0" 06 between the m e m b e r s of a n y pairs of
flasks. The initial p H of the m i x t u r e of starch, diastase a n d water of a series of
eight runs of large flask sampling tests was in the range of 5" 23 to 5" 5. The
range of p H drop from s t a r t to finish in these eight runs averaged 0"09, the
A.G.V. c o n t i n u i n g to rise as hydrolysis proceeded (Table I).

TABLE I

C H A N G E S IN P H A N D S P E K K E R I~EADINGS D U R I N G Two SAMPLING TEST RUNS

Test 1 Test 2
Time After Mixing
Spekker pH Spekker pH
10 minutes 5.35 5.47
20 ,, 5"35-6 5.45
30 ,, 5.33-4 5.44
40 ,, 5.32 5.43
50 ,, 5.32 5.425
60 ,, 5.31-2 5.43
65 ,, 0.38 0"38
70 ,, 5.31 5.43
75 ,, 0.56 0.57
80 ,, 5"31 5" 43
85 ,, 0'69 0.68
90 ,, 5"31 5.43
95 ,, 0-79 0.79
100 ,, 5"31 5.43
105 ,, 0.83 0.84
110 ,, 5.31 5.43

As the statistical e x a m i n a t i o n depended on comparative difference i n

Spekker readings between m e m b e r s of pairs of two control flasks a n d between
m e m b e r s of pairs of control a n d microdose flasks, the results of the series did n o t
depend on d a y - t o - d a y i d e n t i t y of initial p H of the starch-diastase solution
provided the control A.G.V. always fell w i t h i n the p e r m i t t e d range. All solu-
tions on a n y one d a y were made with the one weighing of starch a n d diastase
a n d with the same distilled water a n d were t h u s c o m m o n to all flasks. W i t h the
quantities used the initial p H was always found to be w i t h i n the desired limits.
I n a series of experiments with sets of small flasks, each flask of a set was
i n d i v i d u a l l y a d j u s t e d to a different p H level, using suitable veronal buffer
 EVIDENCE OF THE ACTIVITY OF HIGI~ POTENCIES 15

s o l u t i o n s ( H o b s o n a n d M a c p h e r s o n , 1952). A n i n c r e a s e d q u a n t i t y o f d i a s t a s e
w a s u s e d t o c o m p e n s a t e for t h e s l o w i n g o f t h e r e a c t i o n f r o m t h e use o f t h e buffer.
T h e A . G . V . o f e a c h flask was t a k e n a f t e r n i n e t y m i n u t e s i n c u b a t i o n . I t was
f o u n d t h a t differences o f p H in t h e r a n g e 5"0 t o 5 - 5 g a v e rise t o a difference
b e t w e e n flasks in A . G . V . o f 0 - 0 2 a t m o s t . T w o s u c h sets are s h o w n in F i g . 3.

o.

C4:s-
I
s-o
I
s.s eo
I I
~s
I
~.o
I
~,s
pH REAOI NG

FIG. 3
Two rm~s with small flasks containing starch-diastase solution with in addition veronal
buffer solutions added to give a different p H in each flask. Incubation time ninety minutes
for each flask. The change in velocity of the reaction with change in pH is seen, but it will
be noted that in the range of pH found normally with unbuffered solutions (5.23 to 5.5)
there is no difference in Spokker readings greater than 0.02. Soluble starch 5 g. in 500 ml.
distilled water; O, B.D.H. diastase 0.17 g. in 100 ml. distilled water; • B.D.H. diastase
0" 16 g. in 100 ml. distilled water. Both filtered Whatman 40.

T h i s was w i t h i n t h e p e r m i t t e d e x p e r i m e n t a l v a r i a t i o n a b o u t t h e m e a n o f t h e
c o n t r o l s o f n o r m a l p a i r e d flask t e s t s o n a n y d a y .
I t is d e a r , t h e r e f o r e , t h a t w i t h i n t h e r a n g e o f A . G . V . f o u n d in t h e t e s t s t h e
influence of s m a l l c h a n g e s o f p H w i t h i n 5 - 0 t o 5"5 is negligible. Fig. 4 is a n

.S

,(

o
20
I I I I I I
30 40 50 60 70 80
TiME IN M I N U T E S

FIG. 4.
Sampling test carried out by two technicians to obtain simultaneous pH and A.G.V.
readings.
Soluble starch 5 g. in 500 ml. distilled water. B.D.H. diastase 0.43 g. in 100 ml. dis-
tilled water, filtered Whatman 40, and sintered glass No. 5. Solutions not buffered. Starch
240 ml., diastase 48 ml., and distilled water 48 ml. ~ ~ time at which p H measured.
p H values 5.46 • 0-01 throughout.
16 THE BRITISH HOMG~OPATHIC JOURNAL

example of a sampling test run by two technicians working together so as to

obtain a series of practically simultaneous pH and Spekker values. The arrows
in the figure indicate the times at which pH estimations were made. The pH
values were 5.46 ~=0.01, from thirty minutes incubation onwards.
Further, owing to cross checks in the technique such as pipette interchange,
change in position in the incubation bath etc., there was no reason for properly
cleaned microdose test flasks a l o n e to develop, by chance, pH differences
sufficiently large to alter significantly the rate of hydrolysis. Any such chance
difference, if present at all, would be scattered between control and test pairs
of flasks and would be taken care of by the statistical examination (see also
Appendix D4).
It would appear, therefore: that variations of pH of the order found in
unbuffered tests were insufficient to affect materially the comparative differ-
ences obtained between members of test pairs of flasks.

METHOD
Equipment. The necessary details of the l a b o r a t o r y ventilation w i t h the t e m p e r a t u r e
control a n d of the a p p a r a t u s available for tests arc given in A p p e n d i x B. I n this a p p e n d i x
details are given of the glassware a n d of the pipettes w i t h especial reference to weighing
tests of o u t p u t a n d range of error (Figs. 5, 6, 7).
Pipetting. The 3 ml. vitreosil pipettes for diastase, w a t e r a n d microdose had, on t e n
weighings, an average o u t p u t variation of n o t more t h a n :[_ 0" 002 ml. for a n y pipette, while
the 1 ml. iodine p i p e t t e h a d a m a x i m u m variation of 0. 002 ml., t h u s giving the required
accuracy. The s e m i - a u t o m a t i c suction pipetting is also described.
Incubation. The incubating b a t h , also described, h a d a t e m p e r a t u r e control accuracy of
~=0. 005 a b o u t t h e required t e m p e r a t u r e , which, for crucial tests, was 37.5 ~ C. This gave on
test a t e m p e r a t u r e difference of a p p r o x i m a t e l y 0. 015 ~ C. between the solutions in different
flasks during incubation time (Appendix B).
Cleaning of Glassware. The n a t u r e of the e x p e r i m e n t s requires special cleaning m e t h o d s .
M a n y usual m e t h o d s have been tried a n d discarded. F r o m 1946 to 1951 concentrated H N O a
w a s used for all glassware. Flasks, etc., are n o w cleaned b y Lissapol solutions followed b y
t h o r o u g h rinsing a n d boiling in double distilled water, and b y baking in d r y h e a t a t 150 ~ C.
for 289 hours. Stills are cleaned w i t h concentrated H N O a. P i p e t t e s are t h o r o u g h l y washed,
boiled a n d t h e n baked similarly. Microdose a n d control bottles are treated separately on
similar lines. The p r e s e n t m e t h o d requires t h a t no glassware is allowed to d r y between stages
of cleaning a n d t h a t the only w a t e r coming in contact w i t h t h e m after Lissapol t r e a t m e n t
is distilled. Surgical r u b b e r gloves, free of dusting powder, are used. Full details of the
m e t h o d s of cleaning are given in A p p e n d i x C. A p a r t f r o m insufficient practice in the
technique, lack of a t t e n t i o n to these details is the chief cause of irregular controls.

PREPARATION AND ANALYSIS OF R E A G E N T S

Starch. E x a c t l y 5g. L i n t n e r ' s soluble s t a r c h ( H o p k i n a n d Williams) are weighed on to a
previously weighed p o w d e r paper. This is m a d e into a p a s t e in a small beaker w i t h cold
distilled water. A b o u t 100 ml. of boiling distilled w a t e r is added to the paste f r o m a larger
beaker containing 250 ml. boiling water. The s t a r c h paste a n d boiling w a t e r are t h o r o u g h l y
stirred and transferred to the larger beaker. The small beaker is rinsed w i t h the solution
which is t h e n p o u r e d back into the larger beaker where it boils for three minutes. The
beaker is t h e n r e m o v e d f r o m t h e b u n s e n flame and a b o u t 200 ml. cold w a t e r is added. The
solution is p o u r e d into a 500 ml. g r a d u a t e d flask and the volume is m a d e u p to 500 ml.
w h e n cold. E x a m i n a t i o n of the s t a r c h solution w i t h the d a r k g r o u n d microscope showed it
to be a colloidal solution. This solution which h a d p H = c. 5- 3 gave, w i t h Lugol iodine, a
m a x i m u m a b s o r p t i o n in the region of 600 m ~ on the S p e c t r o p h o t e m e t e r . Culture from
s t a r c h p o w d e r or solution gave a n atypical b. cell (see A p p e n d i x D1 re sterilization).
Diastase. Malt diastase (B.D.H., 1952) is k e p t in a refrigerator. Of this, exactly 0.15 g.
is weighed on to a previously weighed p o w d e r paper, and p u t into a 100 ml. g r a d u a t e d flask
t h r o u g h a filter funnel. Cold distilled w a t e r is added t h r o u g h the funnel to the flask u p to
the g r a d u a t e d m a r k . The flask is s h a k e n until the diastase is dissolved a n d the solution is
filtered t h r o u g h W h a t m a n No. 40 filter p a p e r into a stoppered bottle. This solution has
p H = c. 5.9. The q u a n t i t y of diastase p o w d e r and the time of incubation suitable to
t h e technique (90 m i n u t e s in latest series) h a s to be selected for each n e w s u p p l y b a t c h of
diastase and s t a r c h using a Spekker s t a n d a r d to give a n A.G.V. of a p p r o x i m a t e l y 0" 70
against distilled w a t e r at 1.0 on addition of 1 ml. Lugol iodine (1 in 100). The t h e r m o s t a b i l i t y ,
u n d e r the particular e x p e r i m e n t a l conditions, of the diastase to be used is of flrstimportance
and the m e t h o d for testing this is described u n d e r procedure. Culture f r o m solution gave
b. subtilis with various batches (see Appendix D1 re sterilization).
 Fro. 5
The Biochemical Laboratory.

Fro. 6
The Nivoc Aperiodic Balance.
[r 1). z6
 FIG. 7
The Spekker Absorptiometer.

FIG. 8
The Stills.
 Fzo. 10
Comparison of microdose a n d control on Spekker Absorptiometor.
 FIG. 9
S e m i - a u t o m a t i c pipetting w i t h suction f r o m a w a t e r p u m p . The r u b b e r t u b e ends in a
r u b b e r cap fitting over the pipette s t e m and h a v i n g a small side opening which p e r m i t s
control of the degree of suction b y the technician's finger.
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 17

Iodine. One ml. Lugol iodine is m a d e u p to 100 ml. w i t h cold distilled w a t e r a n d a fresh
solution is m a d e up for each test. The Lugol iodine was m a d e specially w i t h spectrographi-
cally tested distilled water.
Water. The double distilled w a t e r (pyrogen free) for m a k i n g all experimental solutions is
obtained f r o m a Baraglass still followed b y a vitreosil still a n d is the same as t h a t used for
the final stages of cleaning (Appendix C). W a t e r samples are periodically cultured for
bacterial or fungal g r o w t h a n d should be sterile. F r e q u e n t conductivity tests are m a d e to
check the p u r i t y a n d a s t a n d a r d of specific c o n d u c t i v i t y of a p p r o x i m a t e l y 1.5 x 10 -~ m h o s
after s t a n d i n g in a flask is considered suitable. On delivery f r o m the still this w a t e r h a s a p H
o f over 5.8 a n d sp. conductivity of a p p r o x i m a t e l y 0.7 x l0 6 m h o s (Fig. 8).
Mierodoses. The mierodoses are carefully p r e p a r e d a n d are handled in accordance with
certain a s s u m p t i o n s discussed in detail in A p p e n d i x F(B). Twenty-six n u m b e r e d p y r e x
weighing bottles w i t h g r o u n d glass s t o p p e r s chosen at r a n d o m f r o m a pool are placed in
n u m b e r e d holes on a rack. The first one is filled w i t h l0 ml. of double distilled water, while
the remaining 25 are filled w i t h 5 ml. double distilled water, using a vitreosil pipette.
The initial solution is m a d e b y dissolving 0" 1 g. mercuric chloride (Hydrarg. perchlor.
obtained f r o m Nelson) in 10 ml. double distilled w a t e r in the first weighing bottle giving a
solution of 1 in 100 t e r m e d S1. F r o m this p r i m a r y solution one drop is transferred b y
capillary t u b e to the second weighing bottle already containing 5 c.c. double distilled water,
the first bottle being replaced in the rack. The second bottle is succussed ten times after
which one drop of this solution t e r m e d $2 is transferred to the third bottle from the rack
containing 5 ml. double distilled water. The second bottle is t h e n r e t u r n e d to its correct place
in the rack a n d the t h i r d bottle is succussed ten times giving a solution t e r m e d $3. The
same procedure is continued up to solution $26. This solution n o r m a l l y provides the c o m m o n
solution for all the separate microdoses used during a n y one set of experiments, a n d because
succussion is used, the solution is a s s u m e d to be sufficiently homogeneous. The question of
h o m o g e n e i t y a n d dilution is discussed later (Appendix F).
Before m a k i n g the particular microdoses for each d a y ' s test, 5 ml. of t h e control
solutions of double distilled w a t e r are p i p e t t e d into separate weighing bottles which are
placed in separate holes a t the back of the rack. The bottles for the fresh microdoses are
t h e n filled w i t h 5 ml. w a t e r f r o m the same flask using the same pipette a n d placed in a
separate r o w in the rack. This procedure is carried o u t before a n y of the twenty-six
previously p r e p a r e d mierodose solution bottles are touched. The use of a n u m b e r e d rack w i t h
replacement of each bottle after use is essential to avoid unconscious error in the sequence.
The f u r t h e r microdoses for each test are t h e n prepared w i t h these additional bottles. E a c h
n m n b e r e d microdose bottle w i t h g r o u n d glass s t o p p e r is protected from d u s t b y a glass
cover,
The $26 source for test microdoses a l t h o u g h m a d e in distilled water, if p r e p a r e d w i t h
care, k e p t cool a n d protected in a stoppered p y r e x weighing bottle w i t h a glass cover will
r e m a i n " a c t i v e " for some time. Should, however, the t r e n d t o w a r d s s t i m u l a t i o n a p p e a r
to cease, an extremely rare occurrence except w i t h old solutions, then, after two or three
days of negative sixteen flask tests, it is advisable to r e m a k e the microdose series. This
difference of activity w a s we]lillustratedin the "blindfold" series where one set ofmicrodoses
($31) were m a d e from a $26 stage which w a s three m o n t h s old a n d two other sets ($27) f r o m
new $26 stages of t w o f r e s h l y m a d e sets of serial dilutions. The activity of the old micredoses
w a s v e r y m u c h l e s s t h a n t h a t of the fresh microdoses ( T a b l e V I I ) . A n y rise o f s p . c o n d u c t i v i t y of
a mierodose sample above 3 ~tmhos at 18 ~ C. also indicates the advisabilityof m a k i n g a fresh
microdose series.
Analysis of Reagents. Table I I gives the spectrographic analysis of the reagents used in
the crucial tests, including the mercuric chloride used for the mierodoses. Cross comparison
shows t h a t , w i t h the exception of Hg, all o t h e r elements tested for in the mercuric chloride
and detected were also p r e s e n t in one or other of the r e a g e n t s used in the control tests, and
were t h u s e x c l u d e d a s a cause ofeffectin the microdose solutions. The gas analysis of the w a t e r
is given in A p p e n d i x D5.
p H and Solutions. W h e n mixed, the solution of starch, diastase a n d water, or mierodose
in water, should have a p H in the region of 5.2 to 5.5 w i t h o u t buffer.

EXPERIMENTAL I~ROCEDD~E
Crucial Tests. The exact timing of events is vitally important for accurate results.
The starch solution is m a d e first. The diastase solution is m a d e at the s a m e time each
day, i.e. 12.30 p.m. I t is t h e n filtered a n d divided into eight small weighing bottles.
Sixteen n u m b e r e d 50 ml. conical flasks are placed on the stainless steel bench at even
t e m p e r a t u r e , a n d the n u m b e r s noted. This n u m b e r includes the flasks to be used for control
blanks. 15 ml. of the s t a r c h solution are p i p e t t e d into each flask using a single 15 ml. vitreosil
pipette.
Beginning at exactly 12.45 p.m. the flasks are quickly placed in order in t h e incubating
b a t h already at 37- 5 ~ C. followed b y the eight small bottles of diastase. T h u s t h e s t a r c h a n d
diastase solutions are w a r m e d in the b a t h for a similar length of time each d a y before the
pipetting begins. They r e m a i n in the b a t h during the p i p e t t i n g (Fig. 9).
Test .Flasks. W h e n the separate starch a n d diastase solutions have reached b a t h
t e m p e r a t u r e seventy-five m i n u t e s being found necessary for this, 3 ml. of w a t e r are added
3
18 THE BRITISH HOM(EOPATHIC JOURI~AL

TAttLE I I
SPECTROGRAPHIC COMPARISON OF REAGENTS

B a t c h No.
Distilled Soluble
Element Mercuric Chloride 313410/511213
Water Starch
Diastase
p.p.m. p.p.m.
p.p.m. I

Sodium Present 0.1 to 0 . 2 400 200

Potassium N.D. N.D. 2000 80
Phosphorus N.T. N.T. 200 100
Magnesium Present 0.2 400 150
Calcium Present 0.5 300 100
Alumininm N.D. <0.05 100 60
Silicon Present 0.2 40 60
Iron Present 0. 1 to 0.2 20 40
Tin N.D. N.D. < 0.04 20
Manganese N.D. N.D. 2 0.4
Copper Present 0.1 1 0.4
Strontium N.D. N.T. 0.4 0.4
Barium N.D. N.D. <0.2 <0.2
Lead Present N.D. <0.2 0-2
Silver N.D. N.D. <0.1 <0.1
Mercury (Chemically present 74.1 ~ ) N.D. N.D. N.D.
Lithium N.T. N.D. 4 lto2
Rubidium N.D. N.T. < 20 N.D.
Boron* Present <2 <2 N.T.

* I t is difficult to estimate the a m o u n t s as B o r o n is present in the graphite electrodes.

A l u m i n i u m trace in w a t e r doubtful.
N.D. = N o t detected. N.T. = N o t tested for.
E l e m e n t s tested for b u t n o t detected:
Mercuric Chloride ~
Diastase [ Bi, Cd, Co, Cr, As, Au, Be, Ga, Ge, I n , Ni, Te, V, Sb, Zr, W, Zn.
Soluble Starch ]
Distilled W a t e r Bi, Cd, Co, Cr, Ni, Sb, Zn.
Note t h a t Mercuryis the onlyingredient in Mercuric ehloridewhich is not p r e s e n t inone of
the o t h e r reagents. The q u a n t i t y of Mercury found chemically indicates c o n t e n t of over
99 per cent. Mercuric chloride.

t o the first flask, the w a t e r being t a k e n f r o m a flask of the same w a t e r as t h a t used for the
microdoses. E x a c t l y one m i n u t e later 3 ml. of microdose are added to the second flask from
a microdose bottle on the bench. The same pipette is used for w a t e r a n d microdose and it is
t h e n discarded into a vitreosil p o t of distilled water. The n u m b e r s of all pipettes are also
noted.
E x a c t l y two m i n u t e s f r o m the t i m e the second flask received its microdose the first
flask receives 3 ml. of diastase f r o m one of the eight bottles of diastase in the b a t h , a n d one
m i n u t e later t h e second flask receives the same q u a n t i t y f r o m the same bottle delivered
f r o m the same pipette which is t h e n discarded into the same vitreosil pot, being considered
c o n t a m i n a t e d b y the microdose already in the second flask.
F o u r m i n u t e s f r o m the time the second flask begins to receive its diastase the t h i r d a n d
f o u r t h flasks receive their water, microdose a n d diastase in the same w a y as the first and
second, a n d so on. The whole operation is timed so t h a t the first of each pair of flasks receives
its diastase seven m i n u t e s after the second of each preceding pair.
Control Blanks. Along w i t h the test pairs of flasks and included in the time sequence
are either two or four pairs of control blanks, the first flask receiving w a t e r as above a n d the
second also receiving the same w a t e r delivered from the same pipette b u t t a k e n f r o m a small
bottle similar to the microdose bottles.
The pipettes are always w e t w i t h the solution before use. After the solution is r u n into
the flask the surface of the liquid is touched w i t h the tip of the pipette.
A separate small bottle of diastase is used for pipetting for each pair of flasks instead of
d r a w i n g f r o m one large flask in order to p r e v e n t p i p e t t e s which h a v e touched the surface of
the liquid in the first flask f r o m transferring starch-diastase m i x t u r e into a c o m m o n solution.
After each flask has received its three solutions it is gently s h a k e n to m i x t h e m , while
all flasks are s h a k e n gently f r o m time to time during incubation, w i t h o u t r e m o v i n g t h e m
f r o m the b a t h .
E x a c t l y n i n e t y m i n u t e s f r o m the time at which it begins to receive its diastase the first
flask is r e m o v e d f r o m the b a t h a n d is immediately cooled in a t a n k w i t h r u n n i n g t a p water.
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 19

E x a c t l y one m i n u t e later the second flask of the pair is r e m o v e d from the b a t h and p u t in the
cold tank. The first flask is allowed to cool for exactly 289 minutes, w h e n 1 ml. of the Lugol
iodine solution is p i p e t t e d in a n d the flask is shaken.
The a b s o r p t i o m e t e r cell (1 cm.) of the Spekker is rinsed out w i t h the solution f r o m the
first flask a n d is t h e n filled w i t h the solution and p u t into the Spekker. The Spekker is set
at 0-30 on the d r u m and a d j u s t e d to balance w i t h the g a l v a n o m e t e r reading zero. B y t h e
time this has been done t h e second flask has been cooling for 289m i n u t e s a n d exactly at t h e
required time 1 ml. of iodine solution is added to it a n d the flask is shaken. The second cell
is rinsed a n d filled w i t h the solution a n d the A.G.V. of the solution w i t h the s t a r c h iodine
colour is c o m p a r e d w i t h t h e A.G.V. of the previous solution of the pair. The first m e t h o d
gives a c o m p a r a t i v e difference b e t w e e n each second flask and a p r e d e t e r m i n e d s t a n d a r d
zero of 0.30 for the first flask. These results provide the m a i n d a t a for statistical analysis.
The a b s o r p t i o n of the two solutions is t h e n c o m p a r e d w i t h t h a t of distilled w a t e r as a
check on technique. A n o t h e r a b s o r p t i o m e t e r cell is filled w i t h distilled w a t e r a n d the
Spekker a d j u s t e d w i t h this cell to give a g a l v a n o m e t e r reading of zero w i t h a d r u m reading of
1.0: t h e n the t w o cells used for the test are again e x a m i n e d in sequence a n d the reading
noted. This is referred to as the second m e t h o d .
The t e m p e r a t u r e s of the solutions in the cells are t h e n t a k e n as a check against a n y
relative change in a b s o r p t i o n being due to t e m p e r a t u r e change d u r i n g the particular
e x a m i n a t i o n of each pair.
T i m i n g is k e p t accurately t h r o u g h o u t the whole procedure. An electric wall clock w i t h a
large second h a n d is used for reference.
The test is continued thus, each flask h a v i n g exactly n i n e t y m i n u t e s incubation t i m e
and exactly 289m i n u t e s cooling before iodine is added.
The l a b o r a t o r y t e m p e r a t u r e , h u m i d i t y a n d b a r o m e t r i c pressure are noted each day.
The technician wears a head cover a n d a gauze m a s k over the nose a n d m o u t h , w h e n pipet-
ring for crucial tests (Fig. 10).
F r o m d a y to d a y the pipettes for control blanks or microdose tests are interchanged.
F l a s k s are chosen at r a n d o m for t e s t or control pairs of flasks, a n d the position of these
latter in the incubation b a t h is changed f r o m d a y to day, the sequence of control a n d t e s t
pairs being altered each day. A b o u t n i n e t y n u m b e r e d weighing bottles w i t h g r o u n d glass
stoppers, indiscriminately mixed, are k e p t protected f r o m d u s t in a rack. The bottles a r e
picked at r a n d o m , for either microdose or control w a t e r (see reagents). L a t e r it was f o u n d
t h a t a satisfactory p r o p o r t i o n of the bottles h a d been used during the tests for b o t h micro-
dose a n d control (Appendix D4). One set of eight pipettes was kept for diastase only a n d a
second set of eight for either w a t e r or microdose. This m e t h o d of interchange was necessary
to exclude a d s o r p t i o n of mercuric chloride on the glass as an explanation of the results.
I t will be seen f r o m this procedure t h a t the only time variable is the progressively
longer t i m e of preliminary h e a t i n g of the diastase before its addition to the s t a r c h as the
tests proceed. This a m o u n t s to seven m i n u t e s b e t w e e n each successive p a i r of flasks a n d one
m i n u t e b e t w e e n each m e m b e r of a pair. I t is, therefore, of i m p o r t a n c e t h a t the t h e r m o -
stability a t 37.5 ~ C. of the diastase used should be good for the d u r a t i o n of the experiment.
The seven m i n u t e periods m a y , therefore, cause slight variation in values as a g a i n s t
distilled w a t e r for s e p a r a t e pairs of flasks. This does n o t affect the results, as this
time period is the same for b o t h m e m b e r s of each pair, a n d results are concerned solely w i t h
differences b e t w e e n the individual m e m b e r s of each pair. The A.G.V. variation of all the
control flasks, as referred to distilled w a t e r at 1" 0, should n o t be greater t h a n 4- 0.02
d i s t r i b u t e d a b o u t the m e a n reading of the first control flasks of all the pairs for t h a t parti-
cular day. A n y first control flask showing a greater deviation m a k e s the result of t h a t pair
suspect.
The first m e t h o d w h e r e b y the first flask is set at 0.30 a u t o m a t i c a l l y neglects these small
changes b e t w e e n the pairs which do n o t affect the statistics, while the second m e t h o d acts
as a check against a n a b e r r a n t first flask. Such a n a b e r r a n t value, if low, m i g h t m a k e t h e
second flask relatively higher a n d suggest a stimulation. Such a n occurrence was v e r y r a r e
a n d only occurred w i t h the first flasks o f two test pairs o u t of 296, a n d these two results were
accordingly excluded.
W i t h the second m e t h o d , a n alteration in the level of m e a n values of all control flasks
against distilled w a t e r at 1.0 m a y be found on different days. This alteration, which is due
to various experimental factors affecting the overall rate o f reaction, such as h u m i d i t y affect-
ing the a p p a r e n t weight o f s t a r c h or diastase, w a s of no statistical consequence, as all flasks
were involved for the particular day.
W i t h i n this d a y to d a y range o f f r o m a b o u t 0- 60 to 0.80, owing to the design o f the
a b s o r p t i o m e t e r , it was to be expected t h a t a given difference in A.G.V. on different p a r t s of
the scale would be equivalent as, for example, t h a t b e t w e e n 0-60 a n d 0.62 a n d between
0.78 a n d 0.80. I n practice b o t h m e t h o d s were used for all tests a n d the m e a s u r e d differences
b e t w e e n individual m e m b e r s of pairs o f flasks as given b y the first m e t h o d where 0.30 w a s
used as s t a n d a r d for the first flask of each pair a n d the second m e t h o d , were practically
identical w i t h i n 0" 005 on the Spekker (Appendix B). These daily variations in r a t e of
reaction w i t h i n control limits do not, therefore, affect the s u m m a t i o n of Spekker differences
for statistical analysis. W h e r e the readings fell between scale markings, it was possible to
read to a p p r o x i m a t e l y 0. 005, and a n y other indeterminate readings were allotted in the
20 THE BRITISH HOM(EOPATHIC JOURNAL

n o r m a l m a n n e r . I n t h e statistical analysis such readings as fell between the g r o u p interval

used were redistributed in conformity with n o r m a l statistical procedure.
The preliminary b a t h heating t i m e also results in a one m i n u t e difference between the
h e a t i n g time of the diastase for the first flask and the diastase for the second of each pair,
a n y effect on the diastase occurring before the diastase is added to the second flask, as the
actual incubation time for each flask after the addition is exactly the same. The m e t h o d of
c o m p a r i s o n for statistical p u r p o s e s a n d the procedure a d o p t e d p r o v i d e d for this difference
b e t w e e n the first a n d second flasks b y using the first as the relative standard. The statisti-
cal results in the series were, therefore, always d e p e n d e n t on the relative reading of the
second flasks w h e t h e r of control or test pairs, all the pairs being subject to t h e same one
m i n u t e difference. The r e c o m m e n d e d control range of differences of =t= 0' 02 between
m e m b e r s of control pairs distributed a b o u t the setting of 0.30 a d j u s t e d for the first flask of
the p a i r included a n y small difference due to this one m i n u t e difference in preheating.
An e x a m p l e is given of a w o r k i n g record of a single session using this procedure, the
table showing the scattered n a t u r e of the differences obtained (Table I I I ) .

SPECIAI, TESTS FOR UNCONSCIOUSBIAS

T h e procedure for these tests w a s exactly the same, except t h a t the microdose a n d
control bottles were first noted and the result p u t in a n envelope, t h e n the bottle n u m b e r s
were covered b y similar o p a q u e labels. The technician t h e n left the l a b o r a t o r y a n d a n o t h e r
m e m b e r of staff w h o h a d no knowledge of the correct n u m b e r s came in, n u m b e r e d the
fresh labels a n d m i x e d the position of the bottles, after which she left t h e l a b o r a t o r y with-
o u t f u r t h e r contact w i t h the technician. I n the experiment the technician followed the
procedure as if all the bottles w i t h their new n u m b e r s contained microdoses, the control
w a t e r for the first flasks of each pair being t a k e n f r o m a c o m m o n flask.
After the complete test h a d been carried t h r o u g h , the labels were scraped off the bottles
a n d the original code looked up, a n d the results t h u s arrived at.
E x t r a cleaning of the outside of the bottles was required to eliminate a n y traces of
gum. Otherwise t h e interchange of pipettes, flasks, bottles a n d b a t h positions w a s carried
o u t as usual.
E x a m p l e s of this interchange in 1952 are given in A p p e n d i x D4 along w i t h e x a m p l e s
f r o m 1946. A similar f o r m of interchange was used for all tests in the 1952 series, and also in
1946 a n d 1948 except t h a t in these earlier years b o t h the diastase a n d the microdose or w a t e r
p i p e t t e s were also mixed.

SAMPLING TESTS
The s t a r c h a n d diastase solutions are m a d e up in bulk as in the crucial test procedure.
The relative quantities of diastase to s t a r c h are n o r m a l l y the same, b u t for special experi-
m e n t s such as testing the c o m p a r a t i v e activity of samples of diastase, quantities were suit-
ably adjusted. The s t a r c h a n d diastase solutions are m e a s u r e d into s e p a r a t e flasks, 240 ml.
s t a r c h solution into the one a n d 48 ml. diastase plus 48 ml. distilled w a t e r into the other and
b o t h are t h e n placed in the i n c u b a t i n g b a t h a t 37.5 ~ C. a t 12.45 p . m . At 2.30 p.m. the dia-
stase a n d w a t e r solution is added to t h e 500 ml. s t a r c h flask a n d the solutions m i x e d b y
gentle shaking.
After s i x t y m i n u t e s or o t h e r suitable time samples of 21 ml., the a m o u n t n o r m a l l y
used in small flask tests, are w i t h d r a w n a t intervals, usually of t e n m i n u t e s , t r a n s f e r r e d to a
conical flask which is t h e n cooled for 289 m i n u t e s a n d 1 ml. Lugol iodine (1:100) added. A
Spekker cell is t h e n rinsed w i t h the solution a n d filled, the A.G.V. being c o m p a r e d against a
zero for distilled w a t e r at 1.0 (Fig. 1).

TEST FOR THERMo-STABILITY OF DIASTASE DURING EXPERIMENT

A test of sixteen pairs of control blanks is carried o u t n o r m a l l y a n d the A.G.V. against
distilled w a t e r at 1.0 is n o t e d for b o t h f a s k s of each pair. Owing to the seven m i n u t e s
b e t w e e n each pair a n d the fixed incubation time, it is possible to calculate the length of
time during which the diastase for each flask of the pairs has been in solution a n d subjected
to the particular circumstances required for t h e test, the second of each pair being one
m i n u t e longer t h a n t h e first. N o r m a l l y this is the time of preheating at 37.5 ~ added to the
time before the diastase is added to a particular pair of flasks. This preheating period m a y be
at an altered t e m p e r a t u r e or be s u b s t i t u t e d b y a cold b a t h , according to the i n f o r m a t i o n
required.
Fig. 11 shows the partial deactivation w i t h p r e h e a t i n g time a t 37-5 ~ of one b a t c h of
Merck, c o m p a r e d w i t h the b e t t e r t h e r m o s t a b i l i t y of the diastase used for the 1952 tests. The
partial deactivation of the 0~-amylase is also s h o w n (Fig. 12). The reading of the first flask
of each pair is given. This was m u c h more r a p i d a n d increased w i t h dilution of the ~-amylase.
This is a test, n o t for activity against time, b u t to ascertain the effect of preheating on the
diastase w h e n in solution. F o r this, c o m p a r i s o n requires to be m a d e between solutions of
starch-diastase incubated for t h e s a m e time, b u t w i t h different time of preheating.
 EVIDEI~OE OF THE ACTIVITY OF HIGH POTEI~CIES 21

o o o o o o o o o o o o o o o o

r~
C~bO

0 T~

o
9 ,~ o

9 0 0 0 0
++l+

+11+
N~
r~ e'

I++++lll

| ~ ~-~ 0
.B~
"~ "~ ~ ~ .o
9

r ~ ~ -'~

~ ~ ~ . ~
0 0 0 0 0 0
22 THE BRITISH HOME~OPATHIC JOURNAL

I'C

.4 --

'2

I I I I I l I
00 I I0 120 130 140 150 IbO t70

TiME IN M I N U T E S

FIG. iI
Comparative 16 flask tests of batcbes of Merck and B.D.H. diastase for thermostability.
Q u a n t i t i e s of Merck a n d B . D . H . d i a s t a s e were a d j u s t e d to c o m p a r a b l e A.G.V. r e a d i n g s for
e a c h flask a t n i n e t y m i n u t e s i n c u b a t i o n . Soluble s t a r c h 5 g. in 500 ml. distilled w a t e r , x,
Merck d i a s t a s e 0 . 0 5 g.; 9 B . D . H . d i a s t a s e 0 . 1 5 g., b o t h in 100 ml. distilled w a t e r , filtered
W h a t m a n 40. T h e S p e k k e r r e a d i n g of t h e first flask of each pair o f flasks is given. T h e t i m e
i n d i c a t e s t h e period d u r i n g w h i c h t h e d i a s t a s e w a s h e a t e d in t h e i n c u b a t i o n b a t h a t 37" 5 ~ C.
prior to being p i p e t t e d into flasks w i t h s t a r c h solution, after w h i c h i n c u b a t i o n t i m e for all
flasks w a s identical ( n i n e t y m i n u t e s ) . T h e t h e r m o s t a b i l i t y of t h e 1952 b a t c h of B . D . H .
d i a s t a s e is b e t t e r t h a n t h e p a r t i c u l a r b a t c h of Merck diastase. T h e s m a l l e r q u a n t i t y of
Merck r e q u i r e d s h o w s a g r e a t e r a c t i v i t y w i t h g r e a t e r t h e r m o l a b i l i t y .

I-O - -

98 - -
t ----....

o ~

o:

I --

I I I I I I J
lO 20 30 40 50 60 70

TIME IN MINUTES

FIG. 12
C o m p a r a t i v e 16 flask t e s t of m-amylase for t h e r m o s t a b i l i t y . Soluble s t a r c h 5 g. in 500 ml.
distilled water, filtered W h a t m a n 42. 0c-amylase 0-06 g. in 500 ml. distilled water, filtered
W h a t m a n 40. T h e t i m e a m y l a s e w a s k e p t in cold b a t h (14 ~ C.) u n t i l p i p e t t e d into s t a r c h
solution in s e p a r a t e flasks is s h o w n . I n c u b a t i o n t i m e s i x t y m i n u t e s t h e r e a f t e r for all flasks.
T h e degree of dilution of t h e m-amylase r e q u i r e d w a s m u c h g r e a t e r t h a n w i t h t h e o t h e r
d i a s t a s e s tested, s h o w i n g h i g h a c t i v i t y , while in spite of p r e l i m i n a r y cooling a n d s h o r t t i m e
before p i p e t t i n g m a r k e d t h e r m o l a b i l i t y w a s f o u n d . F i r s t flask results.
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 23

ACCESSORY CONTROLPI~OCEDURES
A n u m b e r of additional control procedures were carried out to check or eliminate
factors which f r o m a critical viewpoint m i g h t be t h o u g h t to cause chance variation in t h e
c o m p a r a t i v e readings of test pairs. E a c h is separately dealt w i t h in the A p p e n d i x D.
1. Bacteriological factors in s t a r c h a n d diastase. Possible effect of b. subtilis.
2. Variable factors in the starch-iodine complex, such as t e m p e r a t u r e of solution in
Spekker cells, c o n t i n u a t i o n of hydrolysis during Spekker m e a s u r e m e n t .
3. Effect of mercuric chloride on starch-iodine eolour.
4. I n t e r c h a n g e of glassware. Control exclusion of a d s o r p t i o n f r o m prior molecular
solutions of mercuric chloride as a n explanation of results.
5. Possible c o n t a m i n a t i o n of p r e p a r e d microdoses. Effect of Succussion.
6. Effect of CO~ concentration. Conductivity m e a s u r e m e n t s .
7. Use of Lissapol N in cleaning.

RESULTS
The results obtained were very suitable for statistical analysis of the
differences b y methods widely used in biological experiments where effects are
of the scattered type.
I would like to acknowledge to Dr. R. A. Robb, M.A., F.R.A.S., Lecturer
in Statistics, Glasgow University, m y gratitude for his help and advice on the
statistical problems over the m a n y years of the research work. B y visiting the
laboratories and considering the technique used, he was able to advise statisti-
cians on the best methods for the analysis of the results. The chief method used
throughout has been the Fisher t-test. In addition, the significances of the
variances were tested and where these differed significantly Coehran and Cox's
test was applied as well.
From the statistical point of view, after studying the technique, Dr. Robb
in a personal communication commented "actually your technique, with all the
variables which have beset you, follows the method I have outlined and you have
done everything possible to reduce to a minimum these causes of experimental
error, such as room temperature, pipettes, fatigue etc.".
The statistical results of the tests carried out in 1946 and 1948 are shown in
Table IV. The analysis was made by Mr. J. C. Gunn, M.A., Fellow of St. John's

TABLE I V
ANALYSIS BY PROFESSOR J. C. GUNlV OF RESULTS OF EXPERIMENTS MADE IN JANUARY TO
APRIL, 1946, AND MAY TO JULY, 1948

Corresponding
No. Mean of S u m of
Solution of Mean A b s o r p t i o m e t m Squares t P Remarks
Tests Differences
F
1946
Control 53 0-038 --.00481
$26 26 2.538 +'00769 80.46 / 8.2 <.001 H i g h l y significant
$27 26 2.115 +-00557 60.65 / 7.4 <.001
$28 26 2.077 +.00538 I 63.85/7.2 <.001
$29 25 2.160 +.00580 59.36 / 7 . 5 ~.001
$30 25 1.960 + .00480 46.96 7.2 ~ - 001
Mixed 128 2.172 + "00586 316.4 9.2 ~.001

1948
Control 32 0.688 +.00344 248- 9
$27 32 4-906 + .02453 572.7 4.6 ~.001 H i g h l y significant

Control: b o t h flasks contained distilled water, s t a r c h a n d diastase.

Test: one flask contained distilled water, s t a r c h and diastase. The other flask contained
microdose in distilled water, s t a r c h a n d diastase.
The results were based on an a b s o r p t i o m e t e r reading for the first flask of 9200 in 1946
a n d 9300 in 1948. The group interval was 9005. F o r information, the corresponding m e a n of
the a b s o r p t i o m e t e r differences for each set of results has been added to the Table.
24 THE BRITISH HOM(EOPATItIC JOURNAL

College, Cambridge, now Professor of N a t u r a l P h i l o s o p h y (Cargill Chair),

Glasgow U n i v e r s i t y , to w h o m I would express m y sincere t h a n k s for t h e
s t a t i s t i c a l w o r k he so k i n d l y u n d e r t o o k for t h e T r u s t from t i m e t o t i m e (Boyd,
1942, 1946). Professor G u n n r e p o r t e d on t h e 1946 results: "significant difference
is shown from t h e controls b y e v e r y set o f t h e series. The p r o b a b i l i t i e s are v e r y
strong indeed. This m e a n s t h a t t h e r e is c e r t a i n l y a difference between y o u r
solutions a n d t h e controls", a n d in 1948 "I h a v e c o m p l e t e d t h e calculations of
statistics for t h e d a t a y o u sent me. The " t " value comes o u t a t 4 . 6 3 which w i t h
n -----62 (number of microdoses a n d controls --2) is, of course, h i g h l y significant."
The results o f t h e t h r e e series in 1952 were a n a l y s e d b y Miss CaiTuthers,
B.Sc., now of t h e M a t h e m a t i c a l D e p a r t m e n t , G l a s g o w U n i v e r s i t y , a n d b y Mr.
P. Giles, B.Sc., o f t h e a c t u a r i a l staff of a large insurance c o m p a n y . The m e t h o d s
used are described in t h e r e p o r t o f Mr. Giles. To t h e m I m a k e grateful acknow-
l e d g m e n t . These results are given in t h e following Tables. The s t a t i s t i c a l
m e t h o d used t e s t s for t h e positive, i.e. s t i m u l a t i v e , effect, in t h e t e s t flasks as
a g a i n s t controls. Tables V a n d V I are shown s e p a r a t e l y in o r d e r to give t h e
s t a t i s t i c a l assessment of t h e a c t i o n of two sets of microdoses of different
" p o t e n c i e s " , i.e. stages of shock dilution, with controls carried o u t a t t h e same
time. I n t h e first series of e x p e r i m e n t s a n a l y s e d in Table V t h e $27 stage
of shock dilution o n l y was used.

TABLE V

ANALYSIS BY MISS CARRUTHERS AND MR. GILES OF RESULTS~OF EXPERIMENTS B~ADE IN

MARCH, 1952, T H E R E A D I N G S OF F L A S K D I F F E R E N C E S H A V I N G B E E N O B T A I N E D B Y T w o
M E T H O D S OF A B S O R P T I O M E T E R ~r

Mean of Sum
Solution 1No~" Absorptiometer of t P Remarks
Tests Differences ~uar~

A Control i- 0 00367
$27 -01103 0040 2.7 9005 Very significant
B Control 30 900300 .00353
$27 34 .01015 9 2.6 9006 Very significant

A. Analysis by Miss Carruthers. The absorptiometer readings of differences between

members of each pair of flasks were obtained by adjusting the absorptiometer to give
9300 with the first flask, and then comparing this with the reading obtained with the
second flask. The group interval was 9005. Green filters were used.
B. Analysis by Mr. Giles. The absorptiometer reading for each member of each pair of flasks
was matched against a basic standard obtained with distilled water and an absorptio-
meter setting at 1, and the differences between members of each pair of flasks thus
arrived at. The group interval was 901. Green filters were used.

The results of t w o different m e t h o d s of p r o c e d u r e used in this series for

m e a s u r e m e n t o f A.G.V. differences were e x a m i n e d s e p a r a t e l y , t h e close similar-
i t y of t h e results i n d i c a t i n g t h e a c c u r a c y of S p e k k e r m e a s u r e m e n t which was, for
each m e t h o d , carried o u t on a different p a r t of t h e S p e k k e r scale 9 I t also
p r o v i d e d strong s u p p o r t for t h e technical r e l i a b i l i t y of t h e control a n d t e s t
results. The results were v e r y significant (see section B, Mr. Giles report).
Microdoses of four different stages, $28, $29, $30 a n d S31, were used in t h e
second series a n a l y s e d in Table VI.
I n these p a r t i c u l a r e x p e r i m e n t s , four different solutions were t e s t e d a t
each session along w i t h controls, b u t in v a r y i n g sequence, t h e results being
c o m b i n e d for analysis when t h e series h a d been completed. These results were
v e r y significant. The i n d i v i d u a l stages showed some v a r i a t i o n in level of signifi-
cance.
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 25

TABLE VI

ANALYSIS BY MISS CARRUTHERS~ CHECKED BY MR. GILES, OF RESULTS OF EXPERIMENTS

~[ADE IN APRIL---MAY, 1 9 5 2

:No~. Mean of Sum

Solutions Absorptiometer of P Remarks
Tests Differences Squares

Control --47 900298 9

Mixed $28
$29, $30 [

$31 78 9 9 2"4 9009 Very significant

The absorptiometer readings of differences between members of each pair of flasks were
obtained by adjusting the absorptiometer to give 9300 with the first flask and then compar-
ing this with the reading obtained with the second flask. The group interval was 9005. Green
filters were used.

Table V I I gives the results of a third series, undertaken as a v e r y strict control

on the technique. One set of microdoses of stage $31 derived from a $26 three
m o n t h s old microdose and t w o sets of $27 derived from t w o independent sets of
mierodoses freshly m a d e were tested. I n this third series the technician was una-
ware of the nature of the solutions being tested, whether control or test. Results
were not checked and allotted to the relevant solutions until all the tests for each
day had been run. Particulars of the precautions taken are given under the
section on Procedure. The statistical results of the t w o freshly m a d e sets of
microdoses were v e r y significant, the old microdoses were not significant, while
the total results of all the tests of this s e r i e s t a k e n t o g e t h e r w e r e v e r y s i g n i f i c a n t .

TABLE V I I

/~NALYSIS P A R T L Y B Y MISS CAICRUTHERS A N D B Y MR. GILES OF R E S U L T S OF 'BLINDFOLD ~

EXPERIMENTS TO EXCLUDE UNCONSCIOUS BIAS, MAY TO J U L Y , 1952

1~O. Mean of Suln

Solution of Absorptioneter of i t P Remarks
Tests Differences Squares 1
~ontrol 59 "00008 9 01105 ,

331(3 months old) 27 I

327 (Fresh) I 27 72 .00736 .02310 2- 5 9006 , Very significant
327 (Analar--fresh)] 18 ]
[
The absorptiometer readings of differences between members of each pair of flasks were
obtained by adjusting the absorptiometer to give - 300 with the first flask and then compar-
ing this with the reading obtained with the second flask. The group interval was - 01. Green
filters were used. The $31, an old microdose was statistically inactive (P ~ .15). I t is
included in the results. Both freshly made microdoses from different sources were signifi-
cantly active.

T h e f i n a l T a b l e V I I I g i v e s t h e s t a t i s t i c a l a n a l y s i s o f all t h e 1952 r e s u l t s
t a k e n t o g e t h e r , i n o r d e r t o p r o v i d e t h e d a t a f o r a h i s t o g r a m . T h e t h r e e series
were run practically consecutively between January and July, and could for
t h i s p u r p o s e b e s u m m a t e d . T h e $27 ( A n a l a r ) s i g n i f i c a n t r e s u l t s i n c l u d e d i n
T a b l e V I I w e r e e x c l u d e d f r o m T a b l e V I I I as t h e m e r c u r i c c h l o r i d e w a s o f
different origin from that used originally.
26 THE BRITISH HOM(EOPAq'HIC JOURI~AL

T.~:SLE V i I I
ANALYSIS BY MR. GILES OF ALL 1952 RESULTS TAKEN TOGETHER

~O. Mean of Sum

Solutions of Absorptiometer of t 1~ Remarks
Tests Differences Squares
All Controls 130 .00192 0~-6--76
9 - - - -
All microdoses 166 .00843 i .0358 ~ 4.4 1<10-4 Very highly
, ! significant

The data for this Table include all the 1952 results, except the result obtained with $27
Analar and included in Table VII (see text). The group interval was 901.

REPORT BY ~ R . P. GILES ON STATISTICAL ANALYSIS OF 1952 RESULTS

A. I n each e x p e r i m e n t we h a v e two samples; firstly t h e o b s e r v e d differences
b e t w e e n first a n d second control blanks, a n d secondly t h e differences between
control flasks a n d microdose flasks. Thus a set of nl " c o n t r o l " t e s t s p r o v i d e s nl
differences x,, i ~ 1, 2, -nl, w i t h an a r i t h m e t i c m e a n 2 ~ l S ( x ) , a n d a set o f
nl
n~ microdose t e s t s provides n2 differences x;, i ~ l, 2, n~, with an arith-
m e t i c m e a n ~' 1 S(x'). The significance of t h e difference 2' - - 2 is t e s t e d b y
n~
m e a n s of Fisher~s t - t e s t where
t -- ~'--5c I/n1 n~\

s 2 = S ( x - - 2 ) 2 + S(x' - - 2') 2
n~ + n~ - - 2
N u m b e r of degrees of freedom : n~ + n2 - - 2.
The tables show t h e m e a n values ~, ~' t o g e t h e r w i t h t h e sums of t h e
squares S ( x - - ~ ) 2, S ( x ' - - ~ ' ) 2. Because t h e t e c h n i q u e a n d m e a s u r e m e n t are
i d e n t i c a l for b o t h control a n d t e s t pairs a n e x p e r i m e n t a l bias in t h e m e a s u r e m e n t
o f second flasks will affect k' a n d 9 equally, a n d so t h e r e will be no bias in ~' --2.
The a s s u m p t i o n of n o r m a l i t y i n h e r e n t in t h e a p p l i c a t i o n of t h e t - t e s t was
t e s t e d b y c o m p a r i n g a c t u a l g r o u p frequencies a n d those e x p e c t e d from a n o r m a l
d i s t r i b u t i o n w i t h t h e same m e a n a n d v a r i a n c e as t h e sample. The difference
b e t w e e n actual a n d expected was f o u n d to be always less t h a n twice t h e s t a n d a r d
d e v i a t i o n - - a v e r y s a t i s f a c t o r y result considering t h e size of t h e samples. This
t e s t was a p p l i e d t o t h e d a t a of Table V, for b o t h t h e s a m p l e of controls a n d t h e
s a m p l e o f tests. The e x a m i n a t i o n o f t h e d a t a for Tables VI, V I I a n d V I I I
showed t h a t t h e y were also suitable for t h e a p p l i c a t i o n of t h e t-test.
A second m e t h o d of analysis has also been applied. The significance o f t h e
v a r i a n c e s has been t e s t e d a n d where these variances h a v e differed significantly
Coehran a n d Cox's t e s t has been a p p l i e d to find w h e t h e r t h e r e is a significant
difference b e t w e e n means.
The results o b t a i n e d h a v e been f o u n d in all cases to be as significant as those
shown in t h e Tables for t h e t-test. F o r e x a m p l e , in Table V I t h e results were
f o u n d to be significant on t h e 0.001 significance level used t h e r e for t h e t-test.
B. F o r those who are u n a c c u s t o m e d to s t a t i s t i c a l tests t h e following m a y
be of interest. The t - t e s t begins b y assuming t h a t all t h e samples, i.e. results
o b t a i n e d , come from one n o r m a l group or p o p u l a t i o n , in which case t h e r e will
be no significant difference b e t w e e n t h e control results a n d t h e microdose
results. If, however, we find t h a t t h e differences between t h e m e a n of t h e control
results t e r m e d ~ a n d t h e m e a n of t h e microdose results ~' is large enough
 EVIDEI~CE OF THE ACTIVITY OF HIGH POTENCIES 27

to be significant we cannot retain the assumption that the results are all part of
the one group. We, therefore, conclude that the control results and the
microdose results belong to different groups, i.e. that some factor in the
experiment, in this case the microdose solutions, has produced the difference.
The level of significance is termed P or probability. The smaller the value
of P, the greater is the significance of the result. In biological experiments a
value of P ---- < 0.05 is considered significant, P = < 0"01 may be termed very
significant, and P ~ < 0.001 or less, highly significant. This latter figure means
in ordinary language that it is 1,000 to 1 against these results having been
obtained by chance.

A histogram of the data for Table V I I I is shown (Fig. 13). This is a diagram-
matic method for giving a pictorial representation of differences between the
sets of results, in this case controls and microdoses.

>.
(J
2.2'
uJ
0
Q: .I
la.I
u_
uJ
Z
_I

1-02
i
CONTROL

-.o, .o
.i I;.o, § § +'04 I
0
t--
I POTENCIES.~__Z I
r~'l
0rl
0
~:.2
a.

Fro. 13
H i s t o g r a m of 1952 results used for Table 8, showing the frequency distribution of differences.
:Note w i t h area to the r i g h t t h a t the + 902 block is larger w i t h " p o t e n c i e s " t h a n w i t h controls
while blocks f u r t h e r to r i g h t are only p r e s e n t w i t h " p o t e n c i e s " . The increase to r i g h t indicates
stimulative effects in t h e microdose flasks, The shift of the m e a n is also seen.

In the actual experiments in 1952, it was found that, in the complete series
of 130 control pairs of blanks and 166 test flask pairs, as shown in the histo-
gram, no control results gave an A.G.V. greater than 0" 32 while an appreciable
number of test flasks, considered separately from the statistical assessment
of the frequencies of test and control results as a whole, gave an A.G.V. greater
than 0" 32.
I t would appear that, statistically, the results obtained in these experi-
ments can be stated to be highly significant and indicate that a difference of a
definite "stimulative" character has been demonstrated between the action of
"high potency" microdose solutions and control solutions of distilled water.
28 THE BRITISH t!OM(EOrATHIC JOURNAL

DISCUSSION
To assist in the consideration of the experiments, brief extracts from two
personal communications are given which state clearly the problems presented
by the results.
In the first, after careful consideration of the MSS. of earlier work pub-
fished in 1942, Professor Gowland Hopkins commented: " I f the results obtained
involve some intellectual puzzlement . . . one must not look to errors
in manipulative technique for an e x p l a n a t i o n . . , we ought first to be quite sure
of the adequacy . . . at these dilutions . . . of the method employed. There is no
doubt that the precautions taken in its application seem quite perfect, but is
there nothing inherent in the method itself which in such a region might lead to
uncertainties in spite of these precautions?" The difficulties he then presented
were the problem of controlling conditions governing the intensity of the starch-
iodine colour, the possibility, with unbuffered solutions, of the irregular dis-
tribution of COs from the chemist's respiration affecting the p H and thus
the velocity of the reaction, and the variation in size of colloidal particles with
irregular adsorption of the metal leading to a n0n-uniform distribution. After
developing these points the writer then added "such suggestions would be hyper-
critical were it not that such extreme dilutions were under study, yielding such
a puzzling result" (Hopkins, 1939). Although these comments referred to experi-
ments dealing with dilutions which, though extreme, were yet in the molecular
range, apart from reference to presence of the metal Hg, they still apply. In
the present paper these difficulties have been discussed and the control pro-
cedures described which eliminate uncertainties of the kind he mentions. Details
are to be found under Procedure, pH and use of Buffers, and especially under
Accessory Control Procedures in Appendix D.
The second communication presented another aspect of the problem more
nearly related to the "high potency" microdoses (Gunn, 1946). " I n considering
possible sources of error one can make a well-marked division into two questions
(a) are the solution and control samples presented for comparison genuinely
what they purport to be? and (b) does the instrumental set up and process of
observation provide a quite unbiassed measurement of the properties of the two
kinds of objects, "control-control" and "control-solution" pairs? The second
question involves simply that the process of measurement should be the same in
the two cases. The question has really nothing to do with instrumental errors or
with any sort of error which is equally applicable to both sets of measurements.
In your experimental technique you have obviously succeeded in reducing
such experimental error to a completely acceptable level, so they need not be
further considered". Professor Gunn then pointed out the value of a blindfold
test where the observer was unaware of the actual type of solution, control or
microdose, being measured. He also recommended that a mierodose solution bio-
chemically active should be spectroscopically examined, as, ff the real degree of
"dilution" was as stated, no trace of the original substance would be detected.
These valuable suggestions have been followed. In this paper there has
been described a carefully planned "blindfold" series under Procedure with a
table recording the result (Table VII). This "blindfold" series provided a rigid
test for unconscious bias as it included a set of tests using $31 microdoses derived
from the $26 stage of a three months old set of microdoses and two sets using
$27 microdoses derived from two freshly made sets ofmicrodoses, one derived from
the mercuric chloride normally used and one from an Analar preparation of the
same drug. The old microdoses were found to be statistically inactive while the
fresh microdoses were found to be very significantly active. All results were
included. The spectrographic analysis of the reagents is given in Table I I I under
I~eagents, while the further spectrographic analysis of"high potency" solutions
found to produce stimulation is given in Appendix D5. They show no distinctive
difference from the control solutions as was expected. The detailed method of
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 29

preparing the microdoses in separate stages, thus ensuring the degree of dilution
recorded, is described under Reagents.
The dominant question that remains is the control of the process of dilution.
I t is to be expected t h a t mercuric chloride is adsorbed to Pyrex glass. I t was,
therefore, necessary to determine whether the stimulation produced b y the
microdoses might have been due to solutions which were not so dilute as
calculation would appear to indicate. While this in no way affects the main
investigations into the possible activity of microdoses prepared b y the particular
technique, it is of considerable importance for any conclusion as to the nature of
the solutions showing activity. Could there be introduced into the solutions in
the bottles used, traces of mercuric chloride due to adsorption to the glass on
some previous occasion when more concentrated mierodoses were used, or could
some flasks or pipettes be affected similarly?
The best method to eliminate the chance of effects due to prior adsorption
appearing only in test microdose flasks or bottles and nowhere else is by cross
control and careful interchange of glassware. Pipettes are continuously checked
as one pipette was always used for both members of each pair of flasks. A
detailed analysis of the interchange of glassware has been made and crucial
examples have been given in Appendix D4. This excludes adsorption to the
glassware of mercuric chloride as an explanation of the results.
The efficiency of the cleaning methods was investigated at the Isotope
School, Harwell, by our physical chemist using radio-isotope methods (Appen-
dix G). Using the radio-isotope Hg 2~ a microdose bottle which had contained
a relatively strong solution of 1 : 400 Hg 2~ CI~ for twenty-four hours was cleaned
in accordance with the cleaningprocedure (Appendix C). Samples of water shaken
and left standing in it showed no radio-activity with methods capable of detect-
ing 10-9 g. The cleaning methods were, therefore, capable of removing any HgCl~
loosely adsorbed to Pyrex glass. Further, spectrographic analysis of active $27
microdoses showed no evidence of Hg in the solutions down to 10 -7.
The normal primary mental reaction that the results are due to contamin-
ants has been met by methods of cleaning (Appendix C) and interchange
of glassware and within the limits of modern analytical procedure, by conduc-
tivity measurements, chemical analysis, and by spectrographic analysis, as
described in the text and in Appendix D5. Appendix D6 deals especially with
the problem of C02 contamination.
There remains the suggestion t h a t an unsuspected difference has been
introduced between the controls and the dilutions during the process of dilution.
Apart from straight extraneous contaminants the possibilities would appear to
be t h a t the shock method of dilution increases the gaseous atmospheric con-
tamination in the microdoses, or t h a t it either affects the distilled water or
disturbs the homogeneity of the dilutions. A further suggestion has been made
that surface tension changes due to the solute cause local concentration on the
surface layer or in the bulk of the liquid (Weiser, 1949). This lack of homo-
geneity might lead to a transference of a higher concentration of the drug at each
dilution than is calculated. Thus the dilutions might still be within the molecular
range though outside the range of physical detection.
The suggestion that the results are due to the effect of shock on the dis-
tilled water used for microdoses, and not to the microdose originally present,
has been excluded by a block of highly significant tests in which the second flask
of the control blanks was given, in place of microdose, 3 ml. distilled water from
a bottle to which had been added a drop of distilled water, the bottle being then
succussed. This was the identical process followed in preparing the microdoses
apart from the presence of the microdose itself. These tests are described in
Appendix D6. Confirmatory tests of the same character were also carried out in
1952.
T h e degree of atmospheric contamination was checked by conductivity
30 THE BRITISH HOM(EOPATHIC JOURNAL

measurements and chemical analysis as described in Appendices D5 and 6, while

any effect on p H is discussed under p H and use of Buffers, in the text.
The question of adequate homogeneity of the solutions and the possibility
of transference of unexpected concentrations from one stage to another is
scarcely likely to permit molecular transfer to 27 stages of 1 to 200 dilution,
but this question is also discussed in Appendix F. I t would certainly be a much
simpler explanation ff for any reason a sufficiently high concentration transfer
took place, but there is no evidence t h a t this is so, while analysis of earlier
stages of dilution contra-indicates this theory (Appendix F), as do radio-
isotope experiments (Appendix G).
I t is, therefore, considered that, as controls have excluded other alternatives
if we allow for limitation of modern analysis including radio-isotope methods to
detection within the limits of 10 -9, we are left with a conclusion which m a y be
defined thus:
1. Microdoses derived from mercuric chloride and prepared b y 27 to 31
stages of serial dilution with mechanical shock, nominally of the order of 10 -61
to 10 -71 , were found to accelerate the hydrolysis of starch with diastase.
2. There being no material trace of mercuric chloride nor of contaminants
in the microdoses additional to those normally present in the same distilled
water as is used for controls, and there being highly significant effects obtained
b y these microdose solutions, it is concluded t h a t an active factor derived from
the mercuric chloride accelerates the hydrolysis of starch with diastase. This
stimulative factor is capable of affecting the microdose distilled water, and of
thus transferring its activity to subsequent ultra molecular stages of the "high
potency" microdose preparations. The nature of this factor is unknown.
I n the five crucial test series reported, 326 pairs ()f control-microdose
flasks and 215 pairs of control-control flasks were compared, i.e. over 1,000
solutions, exclud'mg those used for accessory controls and sampling procedures.

ADDENDUM
This unknown "high potency" factor has also recently been demonstrated
with a biological heart rate recorder (Boyd and Eadie, 1952) b y means of a
technique previously developed to test mierodoses of the molecular range down
to 10 -11 (Boyd, 1953). Figs. 14, 15, 16 and 17 show the lay-out of the recording

BLOCK DIAGRAM O F HEART RATE RECORDER

~ ~ CALIBRATOR STABILISO
EPOWER

:OR

MIECHANICAL
ATTACHMENT~-~---
TOHEART

FIO. 14
Block diagram of circuits. Insets show the selection shaping of the 15 c/s frequency compon-
ent of the e.e.g.
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 31

a p p a r a t u s . This t e c h n i q u e is n o w being a p p l i e d t o t e s t i n g of "high p o t e n c i e s " o f

Strophanthus sarmentosus a n d o t h e r drugs on t h e frog heart. I n t h e earlier 1953
p a p e r details are given of t h e p r i m a r y solution of Strophanthus from which t h e
" h i g h p o t e n c i e s " were derived.
I n these l a t t e r e x p e r i m e n t s , to be p u b l i s h e d in full later, definite evidence
has been o b t a i n e d o f t h e action of microdoses in distilled w a t e r of $27 stages
d e r i v e d f r o m Strophanthus sarmentosus as a g a i n s t controls o f t h e same distilled
water. These h a v e been p r e p a r e d from t h e p r i m a r y solution b y t h e m e t h o d
described u n d e r R e a g e n t s with all t h e p r e c a u t i o n s a g a i n s t c o n t a m i n a t i o n as
d e t a i l e d in this p r e s e n t p a p e r a n d appendices. Fig. 18 gives some e x a m p l e s o f
t h e change in h e a r t r a t e a n d e.c.g, which m a y be p r o d u c e d b y this t y p e of dose,
along with t h e n e g a t i v e result of previous a p p l i c a t i o n of controls of distilled
water. The factor is n o t easily d e s t r o y e d a n d in Fig. 19 e x a m p l e s are shown of its
a c t i v i t y using o r d i n a r y $27 microdoses a n d microdoses p r e v i o u s l y e x p o s e d
to b o t h boiling a n d freezing a n d t h e n b r o u g h t to r o o m t e m p e r a t u r e , along w i t h
controls o f distilled w a t e r s u b m i t t e d t o t h e same procedure. Boiling to t h e
e x t e n t shown does n o t d e s t r o y t h e factor b u t m a y i m p a i r its a c t i v i t y . The
a c t i o n of this factor is selective, d e p e n d i n g on t h e i n d i v i d u a l frog s e n s i t i v i t y
to t h e microdose a t t h e t i m e of application.
Table I X shows t h e f r e q u e n c y of h e a r t r a t e responses to control applica-
tions of 0" 06 ml. distilled water, a n d to a p p l i c a t i o n s of 0" 06 ml. $27 Strophan-
thus sarmentosus, r e c o r d e d up t o t h e d a t e of p r e s e n t a t i o n of this p a p e r (March,
1954). More d e t a i l e d analyses a n d s u p p l e m e n t a r y i n f o r m a t i o n will be p u b l i s h e d
later. Of s e v e n t y - o n e frogs t e s t e d with $27 " h i g h p o t e n c y " microdoses 4 9 . 2
p e r cent. showed r a t e responses c o m p a r e d w i t h 2 . 8 p e r cent. showing r a t e
change after distilled water. All distilled w a t e r controls in this series were given
m e c h a n i c a l shock in t h e same w a y as t h e $27 d r u g solutions.

TABLE IX
Total Frogs--i32. Unsuitable Frogs (rate persistently irregular)--not tested--61. Suitable
Frogs (rate regular prior to drug tests)--tested--71.

Tested Frogs Distilled Water $27 Strophanthus sarmentosus

~o. % No. o/
/o
Frogs reacting* . . . . . . 2"8 35 49-2
Total applications (all frogs tested) 262
Applications (immediate reaction) Nil 0 50~ 19.0
Applications (reaction within 2
minutes) . . . . . . . . 2 0"73 38 14.5
Total applications producing re-
action . . . . . . 2 0"73 88 33"5

* One frog reacted after distilled water only. The other which reacted also responded to
microdose.
Confirmed by rate measurement on simultaneous e.e.g, films except in one ease.
NOTE: $27 equivalent to homceopathic 32c high potency.
The comparative heart rate response of exposed hearts of suitable frogs (Rana Temper-
aria) to direct applications on the auriculo-ventricular junction of 0" 06 ml. double distilled
water control and of microdoses of 0.06 ml. $27 Strophanthus sarmentosus in the same
distilled water. Suitable frogs were those with an approximately regular heart rate recording
after recovery from initial shock and prior to control or drug application.

T h e t o t a l n u m b e r of a p p l i c a t i o n s of distilled w a t e r t o t h e 71 frogs, includ-

ing t h e 36 insensitive frogs, was 274, while t h e $27 microdoses were also a p p l i e d
on 262 occasions. The drug a p p l i c a t i o n s p r o d u c e d 88 r a t e changes (33" 5 p e r
cent.) c o m p a r e d w i t h two r a t e changes occurring after distilled w a t e r (0"73
p e r cent). A n i m m e d i a t e d i s t i n c t i v e r e a c t i o n on a p p l i c a t i o n was o b t a i n e d in
19 p e r cent. of t h e d r u g applications, while no i m m e d i a t e response was f o u n d
32 THE BRITISH ttOM(EOPATttIC JOURNAL

after any distilled water applications. The total number of drug applications to
t h e 35 f r o g s w h i c h s h o w e d a r a t e r e a c t i o n w a s f o u n d t o h a v e b e e n 156 o f w h i c h
56 p e r c e n t . p r o d u c e d a n effect. T h e p r o p o r t i o n o f f r o g p r e p a r a t i o n s f o u n d u n -
suitable for testing owing to persistent irregularity of heart rate was found to
v a r y, increasing in w a r m e r w e a t h e r , a n d at s p a w n i n g time, a n d d i m i n i s h i n g
d u r i n g c o l d e r spells.
It would appear, therefore, that the preparation of solutions derived from a
p r i m a r y s u b s t a n c e b y t h e p h a r m a c e u t i c a l m e t h o d o f serial d i l u t i o n i n s m a l l
q u a n t i t i e s w i t h m e c h a n i c a l shock results in t h e presence o f a selective d e r i v e d
f a c t o r w h i c h is c a p a b l e o f i n f l u e n c i n g b i o c h e m i c a l a c t i v i t y u n d e r s u i t a b l e
experimental conditions.
F u r t h e r i n v e s t i g a t i o n o f t h i s f a c t o r is b e i n g c a r r i e d o u t .

ACKNOWLEDGMENTS
Sincere thanks are due to all chemists and technicians who have taken part in this
research, and to those members of the staff of Glasgow University, of Glasgow Royal
Technical College, of the Rowett Research Institute, and of many other research organiza-
tions who have so generously given advice and information. Grateful thanks are also due to
the physical chemists who have carefully checked the text, and especially to Dr. J. B.
Fettigrew, B.Sc., Consultant Biochemist, Law Hospital, who developed the technique in the
.early years, and who has carefully studied and advised on the text of this report on the
completion of the fifteen year's work. Financial support has been received from the Belt
Research Committee of the British Homceopathic Association, from the Scottish ttomceo-
pathic Research and Educational Trust, and from private subscribers through the Trustees
~)f the Boyd Medical Research Trust, to all of whom grateful acknowledgment is made. In
addition support is being given by the National Health Service Management Board of the
~Glasgow ttomceopathic Hospital for the biological investigation.

APPENDIX A
HYDROLYSIS OF STARCH W I T H DIASTASE
Starch consists essentially of amylose and amylopectin.
Amylose is mainly composed of l:4-a-linked glucose units, but also contains a few
l:3-fl-links (Fig. 20a, b, c). The whole molecule contains 200-300 glucose units, and is
probably a long coiled, but unbranched or only slightly branched, structure.
Amylopectin is extensively branched and is built up from unit chains each containing
~n average 20-24 l:4-~-linked glucose units. An unknown number of these chains are joined
together. I t is not known how inter-unit chain links are arranged except t h a t the terminal
reducing unit of one chain is united to a hydroxyl of a glucose unit on an adjacent c h a i n - -
probably as a rule in the 6-position--i.e. junction of chains made by a l:6-a-link (Baldwin,
1952). Amylopectin is usually represented as a laminated structure (Fig. 20d)(Haworth,
Hirst and Isherwood, 1937).
Lintner's soluble starch was used. This starch, which has been solubilized by the action
.of acids, is no longer capable of swelling, but on treatment with hot water the granules dis-
integrate and pass into solution. The acid treatment breaks the starch down to molecules
of smaller size which are incapable of forming the giant networks characteristic of the
.swollen granules (French, 1950). This starch does not readily retrograde and is the most suit-
able form of starch for enzyme work. I t can be uniformly dispersed by boiling for two
minutes.
Diastase consists essentially of a- and fl-amylases. The diastases are not simple enzymes.
Malt diastase, as used, was free from intentional loading and normally has a large pre-
ponderance of fl-amylase over a-amylase (Bailey, Hopkins and Dolby, 1937). I t also has a
small proportion of other enzymes such as Maltase, R-enzyme and Z-enzyme. These amy-
]ases are either protein entities or are specific groupings intimately associated with specific
proteins. Their properties are typical of proteins. Evidence appears to favour the view that
~he amylases are protein entities and not dependent on prosthetic group for activity
(Kneen, 1950).
fl-amylase is specific for l:4-a-links. Pure fl-amylase catalyses a 70 per cent. conversion
,of amylose to maltose, but complete conversion requires the assistance of the Z-enzyme
which acts on the l:3-fl-glucosidic links. Similarly with amylopectin, fl-amylase acts until a
branching llnk (l:6-~-link) is approached. In this way about 58 per cent. is converted to
maltose. The residual resistant product the so-called a-amylodextrin or "limit dextrin"
gives a port wine colour with iodine. This dextrin can be attacked b y a-amylase which
splits it into smaller fragments which are still of a dextrin-like nature, but no longer give
colour with iodine. Fission of these requires the presence of the recently discovered hydro-
lytic enzyme--R-enzyme, which attacks l:6-g-links.
 [By king permission of Electronic Engineering
FIG. 15
General view of a p p a r a t u s .

FIG. 16
View of the mechanical recorder a t t a c h e d to the apex of the heart. The set-up on the frog
of the R i n g e r - p l a t i n u m electrodes and the Ringer drip s u p p l y is seen. The screening cage,
pre-amplifier, calibrator a n d m a r k i n g trigger are also shown.

[I~ce p. 32
 FIG. 17
Addition of solutions to the auriculo-ventricular junction of the frog hoart using a
calibrated capillary tube.
(a) C o n t i n u o u s record of t h e h e a r t r a t e s h o w i n g t h e r a t e c h a n g e s following five a p p l i c a t i o n s
of t h e $27 w i t h control a p p l i c a t i o n s of 0 . 0 6 ml. of t h e distilled water. E.c.g. o m i t t e d .

(b) A n o t h e r frog s h o w i n g h e a r t r a t e c h a n g e following e a c h of t h r e e a p p l i c a t i o n s of 0 . 0 6 ml.

$27 of t h e s a m e drug. E.e.g. o m i t t e d .

$2~7 ]3 mins. later

(c) T h e e.c.g, r e s p o n s e of a n o t h e r frog h e a r t to, (1) a p p l i c a t i o n of 0 . 0 6 ml. of distilled water,
a n d (2) effect following a p p l i c a t i o n of 0 . 0 6 m]. of $27 m i c r o d o s e of Strophanthus sarmen-
tosus. N o t e t h e m a r k e d depression of P - Q w i t h rise of S-T. (3) L a t e r recovery. T h e c h a n g e
in t h e c o m p l e x a t m o m e n t of a p p l i c a t i o n w a s n o t c o u n t e d as a n effect. R a t e record o m i t t e d .
FIG. 18
Effect of direct applications, on t h e e x p o s e d h e a r t s of t h r e e sensitive frogs, of 0" 06 ml. of $27
microdoses derived f r o m Strophanth~s sarmentosus. $27 m i c r o d o s e s were p r e p a r e d as in t h e
biochemical e x p e r i m e n t s , w i t h sterile p r e c a u t i o n s , in s e p a r a t e bottles b y serial dilution a n d
m e c h a n i c a l shock, t h e d i l u e n t being f r o m t h e s a m e flask of distilled w a t e r as t h a t u s e d for
control applications.
T h e r a t e recorder vertical t i m i n g lines were five m i n u t e s a p a r t . T h e h o r i z o n t a l lines 0 . 4
40 b e a t s p e r m i n u t e etc. T h e m o m e n t of d r u g a p p l i c a t i o n in (a) a n d (b) is i n d i c a t e d b y a
vertical e x c u r s i o n of t h e p e n p r o d u c e d b y a h a n d trigger w h i c h also m a r k s a s i m u l t a n e o u s
e.c.g. T h i s m a r k i n g is also seen in Fig. 19.
(a) R e s p o n s e to 827 Stvophanthus sarmentosus w h e r e an o r d i n a r y m i c r o d o s e w a s a l t e r n a t e d
w i t h a boiled mierodose. T h e c o n t r o l d i s t i l l e d w a t e r in t h e latt)er case w a s also boiled. N o t e
t h e m i c r o d o s e w a s a c t i v e in s p i t e of b e i n g boiled for t e n m i n u t e s a n d a l l o w e d t o cool to
room temperature.

(b) The effect of five doses of m i c r o d o s e $27 Strophanthus sarmentosus, t w o m i c r o d o s e s

h a v i n g been boiled, t h e c o n t r o l d i s t i l l e d w a t e r also h a v i n g be e n boiled. B o t h w e re a l l ow e d t o
cool to r o o m t e m p e r a t u r e . The t w o o t h e r m i c r o d o s e s we re frozen a l ong w i t h t h e d i s t i l l e d
w a t e r c o n t r o l a n d were t h e n a l l o w e d to m e l t a n d come to r o o m t e m p e r a t u r e . I t c a n be seen
t h a t b o t h frozen m i c r o d o s e s were a c t i v e while one b o i l e d m i c r o d o s e w a s a c t i v e a n d t h e
o t h e r o n l y s l i g h t l y a c t i v e . One o r d i n a r y $27 m i c r o d o s e effect w a s i n c l u d e d w i t h i t s d i s t i l l e d
w a t e r control for c o m p a r i s o n .

(c) T he effect of t w o doses of m i c r o d o s e $27 Strophanthus sarmentos~s. The first mi c rodos e ,

a n o r d i n a r y one, g a v e a s u d d e n m a r k e d a g g r a v a t i o n l a s t i n g for a c o n s i d e r a b l e t i m e . The
second microdose, boiled for t e n seconds a n d a l l o w e d to cool to r o o m t e m p e r a t u r e , still
g a v e a definite effect, b u t in t h i s frog to a lesser degree.
FIG. 19
Effeets of p o t e n c i e s w h i c h h a v e b e e n boi l e d a n d frozen.
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 33

(a) (b) (c)

CH2OH CH2OH 6 CH~OH

C C C
~ H

4 I
\ /
!/\. I\o. ! / ~\ o.
c ' 3 2

(d)
F i e . 20
(a) ~--glucose. (b) fl--glucose. (c) Notation.
(d) D i a g r a m m a t i c I4epresentation of L a m i n a t e d Structure o f A m y l o p e e t i n ( H a w o r t h etal.).

~-amylase is also specific for l:4-~-links, breaking amylase down into s u b s t a n c e s of

small moleculax weight. I t can b y - p a s s the branching links of axnylopectin, and if the
reaction is allowed to go to completion gives as a m a i n p r o d u c t maltose a n d some 1:6-
1inked sugars (Baldwin, 1952). I t is principally the s t a r c h liquifying a n d dextrinizing enzyme
while on the average 85 per cent. of the saccharifying activity of the m a l t diastase is due to
fl-amyla.se a n d 15 per cent. to ~-amylasc.

API'ENI)IX B
L A B O R A T O R Y EQUIPMEI~'T F O R T E S T S
Laboratory. The l a b o r a t o r y is k e p t in cold w e a t h e r at an average t e m p e r a t u r e of 18 ~ C.,
t h e r m o s t a t i c a l l y controlled. The air is filtered on e n t r y and extracted b y fans. There are
cooling axrangoments for w a r m e r weather. Stainless steel benches give a surface of even
t e m p e r a t u r e . The lighting w a s u n i f o r m and nor too bright. Lighting producing ultra-violet
radiation m u s t be avoided during experiments, a n d " p o t e n c i e s " protected against such radia-
tion.
Plates exposed to the air of the l a b o r a t o r y showed on culture airborne m o u l d s a n d some
coliform and coccal forms, b u t no b. subtili~.
Heating Over~s'. Two ovens are available, one thermostatically controlled to 150 ~ C., the
other a furnace controlled to 1,000 ~ C.
Incubating Bath. A suitably designed electric m o t o r stirrer is used in the lagged incubat-
ing bath. This m o t o r is separately m o u n t e d on a wall bracket to avoid vibration. The heat-
ing element is thermostatically controlled b y a mercury-toluol control w i t h a sunvic bi-
metal variable head for the needle contacting the mercury. This a r r a n g e m e n t gives a
control accuracy for the selected b a t h T e of 4- 0. 005 ~ C. Care m u s t be taken t<) avoid any
side displacement of the fine needlepoint contacting the m e r c u r y in the control.This degree
of control gave on test an average T e difference of 0-015 ~ C. between the solutions bl
different flasks during the incubation time.
Glassware. Containers, flasks etc. are m a d e either of p y r e x or vitreosil w i t h ground
glass stoppers. Pipettes, except for the iodine pipette, arc m a d e of vitreosil. Boiling t r o u g h s
are of vitreosil. All pipettes, bottles a n d flasks are e n g r a v e d with n u m b e r s to p e r m i t of
interchange in their use for control and microdose.
Pipettes. The n u m b e r e d vitreosil pipctt~s originally tested N.P.L. are checked at
intervals b y ten weighings of o u t p u t using b o t h w a t e r and diastase, the weight of o u t p u t
being m e a s u r e d after t o u c h i n g the solution with the tip of the pipette. Those showing
greatest constancy in o u t p u t and nearest to each other in weight of o u t p u t axe k e p t solely for
the water, microdose a n d diastase. Suction pipetting is used to avoid m o u t h or b r e a t h
c o n t a m i n a t i o n . This pipetting is obtained b y suctiou from a w a t e r p u m p ; the r u b b e r t u b i n g
to the p i p e t t e ends in a r u b b e r cap with a side-opening which allows control b y the finger of
the rate of suction. This cap can be rapidly flicked off the end of the pipette as the o p e r a t o r ' s
first finger is slipped over the end of the pipette. This gives accurate control of meniscus
readings. This semi-automatic m e t h o d was found by weighing to be m u c h m o r e a~curate
t h a n the use of completely a u t o m a t i c pipettes and allowed of rapid changing of pipettes mu
required.
The eight pipettes used for diastase, one for each test or control pair of flasks, were
the m o s t vital, as e x p e r i m e n t a l accuracy could be em~ily affected b y small differences in
q u a n t i t y of diastase. Tests showed t h a t a difference of a p p r o x i m a t e l y 4- 0.04 ml. or m o r e of
34 THE BRITISH HOM(EOPATHIC JOURI~AL

diastase in the second m e m b e r of a pair of flasks m i g h t cause a Spekker reading change

statistically biassed t o w a r d s stimulation. Weighing tests w i t h a l~ivoc Aperiodic Balance
showed, on t e n weighings for each of these p i p e t t e s using diastase, an average o u t p u t
variation of • O. 002 for a n y pipette. The m a x i m u m p i p e t t e error w a s only in one instance
as great as 0. 007. As one diastase p i p e t t e is used for each pair of flasks a n y chance error
between the t w o flasks w a s well w i t h i n the required accuracy.
The average m e a n o u t p u t ef all the pipettes t a k e n t o g e t h e r (80 weighings) w a s 3.00 ml.
• ml. at N . T . P . This m i g h t produce a fractional difference b e t w e e n the value o f
Spekker reading a g a i n s t distilled w a t e r at 1.0 as b e t w e e n different pairs of flasks owing
to the use of a fresh p i p e t t e for each pair, b u t this variation w a s well w i t h i n the p e r m i t t e d
control variation a n d affected b o t h the flasks equally. The first m e t h o d of procedure already
described excluded this factor. The p i p e t t e s used for either w a t e r or microdose had the s a m e
order of accuracy.
Iodine Pipette. The 1 ml. p y r e x p i p e t t e used for 1:100 Lugol solution w a s also vital to
c o n s t a n c y of t h e Spekker reading. The m a x i m u m variation w i t h this p i p e t t e w a s f o u n d to be
0. 002 ml. again well w i t h i n the required accuracy.
Absorptiometer. I n 1946 an old t y p e of Spekker a b s o r p t i o m e t e r w a s used. The 1948 a n d
1952 results were o b t a i n e d w i t h a newer type, 1-I454, w i t h a wider scale and Cambridge s p o t
g a l v a n o m e t e r w i t h shielded scale. This i n s t r u m e n t w a s p u t t h r o u g h a long series of tests w i t h
colour solutions b y an optical physicist and the limits of experimental error a n d best sen-
sitivity setting arrived at. W h e n e v e r a n y signs of increase of this error a p p e a r n e w P . E .
ceils are obtained a n d the performance checked. Two additional filters of Chance 019 glass
to reduce h e a t effects were added, the filter b e t w e e n the lamp casing a n d the c o m p a r i s o n
cells being m a d e sufficiently large to shield all t h e cells f r o m the l a m p casing. The i n s t r u m e n t
is k e p t in a fixed position a w a y f r o m s t r o n g direct light. Set H455 OG1 green filters were
used. The best e x p e r i m e n t a l range of iodine colour for c o m p a r i s o n w i t h green light used w a s
arrived at b y a large n u m b e r of tests, a n d lay between 0.60 a n d 0.80 against distilled w a t e r
a t 1.0, the quantities of diastase being a d j u s t e d accordingly. The Spekker design w i t h
logarithmic scale gives equivalent values for equal numerical scale differences over a n y p a r t
of the scale. The m a r k i n g s on the scale are at 0" 01 intervals f r o m zero to 0- 6, a n d at 0.02
f r o m 0 . 6 to 1.
Cambridge pH Meter. This m e t e r reads pI-I accurately to 0.02. The wide v a r i a t i o n of
m a i n s voltage affected accuracy even w i t h a c o n s t a n t voltage t r a n s f o r m e r . A c c u m u l a t o r s
were s u b s t i t u t e d b u t require to be v e r y carefully checked and discharged to the flat voltage
level before use. A M o r t o n glass electrode cell is used.
Mullard Conductivity Meter. Conductivity m e a s u r e m e n t s are m a d e n o r m a l l y w i t h this
meter, w i t h the Mullard s t a n d a r d b r i g h t a n d black p l a t i n u m cells. The former was used for
solutions of low conductivity, the latter for those of higher conductivity. F o r measure-
m e n t s of w a t e r or microdose solutions in weighing bottles a micro cell w a s designed w i t h
p l a t i n u m bead electrodes and perforated glass collar to ensure c o n s t a n c y of distance f r o m
electrodes to glass. All cell c o n s t a n t s are f r e q u e n t l y checked.
Spectrophotometer. A small U n i c a m S.P. 350 i n s t r u m e n t is used for b r o a d l y m e a s u r i n g
the a b s o r p t i o n of starch-iodine solutions, and of the general iodine complex colour after
incubation of starch-diastase. F o r actual test c o m p a r i s o n of flask colours after incubation,
the a b s o r p t i o m e t e r is m u c h m o r e sensitive a n d accurate.
Succussion Apparatus. A simple a p p a r a t u s is used consisting of a b l o c k of wood carry-
ing a m e t a l container for holding the weighing bottle w i t h the microdose solution to be
subjected to mechanical shock. The block is raised to a fixed height a n d allowed to fall b y
gravity, accelerated b y the tension of a r u b b e r s p r i n g on to h a r d r u b b e r balls.

APPENDIX C
M E T H O D O F CLEA-R!ING G L A S S W A R E F O R 1952 C R U C I A L T E S T S
Water. Double distilled w a t e r is used for all cleaning unless where otherwise stated.
Flasks. After use, flasks are t h o r o u g h l y w a s h e d u n d e r r u n n i n g t a p water, rinsed w i t h
distilled water, a n d placed in a p y r e x t a n k containing a solution of Lissapol (3 t a b l e s p o o n s
Lissapol in 1 gallon distilled water). The flasks are allowed to soak in the solution overnight.
A s e p a r a t e r u b b e r glove is k e p t specially for changing the glassware in a n d o u t of the clean-
ing solution. The s t o p p e r s are also soaked in Lissapol at the s a m e time.
I n t h e m o r n i n g the flasks and s t o p p e r s are r e m o v e d f r o m the Lissapol a n d t h o r o u g h l y
rinsed w i t h once distilled w a t e r to r e m o v e m o s t of the Lissapol, w h e n t h e y are placed in
a n o t h e r t a n k (pyrex), containing double distilled water, and allowed to r e m a i n there for
q u a r t e r of an hour.
Surgical r u b b e r gloves boiled at intervals a n d rinsed in distilled w a t e r before use are
used for handling the glassware for the remaining stages of cleaning. T h e y are k e p t in a glass
container w h e n n o t in use.
T h e flasks are always placed, b e t w e e n cleaning stages, on a strip of glass covered w i t h
sterile gauze. E a c h flask is half-filled w i t h double distilled w a t e r a n d stoppered a n d is t h e n
s h a k e n for a full m i n u t e - - t i m e d exactly b y a clock, after which the s t o p p e r is released
slightly a n d the w a t e r allowed to r u n slowly p a s t the s t o p p e r until the flask is e m p t y . This
w a s h e s b o t h s t o p p e r a n d g r o u n d neck of flask.
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 35

E a c h flask t h e n receives a fresh lot of distilled w a t e r a n d is similarly s h a k e n again for a

full m i n u t e . D u r i n g the s h a k i n g the s t o p p e r is p a r t l y released to allow the w a t e r to reach t h e
neck of t h e flask. After the second s h a k i n g a n d e m p t y i n g , the flasks are filled w i t h fresh
distilled w a t e r a n d placed on their sides in a q u a r t z t r o u g h sufficiently filled w i t h distilled
w a t e r a n d boiled for t w e n t y minutes. The s t o p p e r s are also boiled for t w e n t y m i n u t e s .
E a c h flask is t h e n r e m o v e d f r o m the t r o u g h , emptied and half-filled w i t h w a t e r ,
s t o p p e r e d a n d s h a k e n again for a m i n u t e as previously described. All the flasks are t h e n
e m p t i e d a n d placed upside d o w n in a draining rack a n d allowed to drain u n d e r cover o f
clean white lint. T h e y are t h e n p u t into an oven at 150 ~ C. for 2~ hours. The stoppers, in a
clean beaker, are similarly heated. W h e n cool, the flasks are r e m o v e d f r o m the oven,
s t o p p e r e d a n d placed in a d u s t excluding c u p b o a r d w i t h glass covers over t h e m .
D u r i n g t h e whole cleaning process no piece of glassware is t o u c h e d w i t h the bare h a n d .
Pipettes. After use the control a n d test pipettes, all of vitreosil, are placed in t w o vitreo-
sil p o t s of distilled water. One p o t receives p i p e t t e s which h a v e on t h a t d a y been used for
pipetting microdoses a n d those diastase pipettes w h i c h h a v e contacted the solution of
flasks w i t h microdose. The o t h e r receives pipettes which h a v e n o t been in c o n t a c t w i t h
microd0ses, including the vitreosil s t a r c h a n d the p y r e x iodine pipettes. The p i p e t t e s are
allowed to soak in the p o t s overnight.
R u b b e r gloves are used w h e n cleaning the p i p e t t e s n e x t day. E a c h p i p e t t e is r e m o v e d
f r o m the p o t a n d drained. I t is t h e n t h o r o u g h l y w a s h e d inside and outside w i t h distilled
w a t e r f r o m a w a s h bottle w i t h a pressure bulb. After washing, using a piece of r u b b e r t u b -
ing, w i t h a glass m o u t h p i e c e attached, w a t e r is d r a w n u p the pipette to fill it. I t is immedi-
ately p u t in a vitreosil t r o u g h which has been half-filled w i t h distilled water. All the pipettes,
t r e a t e d in this w a y , are boiled for t w e n t y m i n u t e s , t h e n s h a k e n a n d p u t in the oven w i t h t h e
tips resting on thick sterile gauze and h e a t e d for 289 h o u r s at 150 ~ C.
W h e n cool, t h e y are placed in the special d u s t p r o o f b o x until required. The b o x h a s a
rack w i t h s e p a r a t e n u m b e r e d holes for each pipette.
Filter funnels, graduated flasks, beakers, reagent bottles. After use these are well w a s h e d
u n d e r r u n n i n g t a p water, rinsed w i t h distilled water, a n d placed in t h e Lissapol t a n k over-
night.
I n the m o r n i n g t h e y are r e m o v e d f r o m the Lissapol a n d rinsed w i t h once distilled w a t e r
until all traces of Lissapol " s o a p i n e s s " h a v e disappeared w h e n t h e y are placed in a t a n k
containing distilled w a t e r a n d allowed to soak for q u a r t e r of an hour.
E a c h piece of glassware is t h e n half-filled w i t h w a t e r a n d s h a k e n t h o r o u g h l y for t w o
m i n u t e s , t h e s t o p p e r s of flasks or bottles being released as previously described. After
being e m p t i e d it is refilled a n d s h a k e n for a second time for 2 minutes. After a third rinsing
a n d s h a k i n g the piece of glassware is e m p t i e d a n d placed upside d o w n on a draining b o a r d
a n d allowed to drain for one hour. Thereafter it is heated in the oven for 289h o u r s at 150 ~ C.
W h e n cool it is p u t in the d u s t p r o o f cupboard.
Microdose Bottles. These bottles, except the first ten used to p r e p a r e a " p o t e n c y " , are
rinsed o u t t h o r o u g h l y w i t h distilled w a t e r after use and p u t t h r o u g h Lissapol a n d rinsed w i t h
distilled w a t e r in the s a m e w a y as the rest of the glassware. The flask used for p o u r i n g
distilled w a t e r into microdose bottles is k e p t well a w a y f r o m t h e m - - a t least 6 inches. The
first t e n bottles are e m p t i e d directly d o w n the sink drain, a n d boiled in distilled w a t e r in a
stainless steel basin for t w e n t y m i n u t e s before being p u t into Lissapol. All the bottles are
t h e n placed in a stainless steel basin a n d boiled for t w e n t y minutes. T h e y are picked o u t
w i t h forceps, rinsed w i t h distilled w a t e r a n d placed in a p y r e x dish where t h e y are boiled
again f o r t w e n t y m i n u t e s . The w a t e r is r u n off a n d the bottles boiled again for t w e n t y
m i n u t e s in fresh distilled water. After this t h i r d boiling, bottles and s t o p p e r s (which h a v e
been boiled w i t h the bottles) are r e m o v e d f r o m the dish and p u t into the oven where t h e y are
h e a t e d for 289h o u r s at 150 ~ C.
Capillary Tubes. These arc discarded after use. N e w t u b e s (B.D.It.) arc boiled in a
beaker a n d heated in the oven for 289h o u r s at 150 ~ C.
Spatulas. The s p a t u l a s used for s t a r c h a n d diastase are wiped w i t h a sterile s w a b a n d
boiled for t w e n t y m i n u t e s . T h e y are t h e n p u t in the oven until dry.
Troughs and Vitreosil Dishes. These are heated in the electric furnace until t h e y are red-
hot---a t e m p e r a t u r e of a b o u t 400 ~ C. This is done after the d a y ' s cleaning.
The w a t e r used for all stages of cleaning, after Lissapol t r e a t m e n t , is f r o m a Baraglass
still followed b y a vitreosil still. The intermediate side a r m of the vitreosil still is k e p t
p e r m a n e n t l y covered w i t h sterile silk w a t e r p r o o f tissue t h r o u g h which passes the outlet t u b e
of the Baraglass still. All containers are either stoppered or k e p t covered w i t h a plastic
a n d sterile gauze cover. The receiving flask has a p y r e x cap w i t h a hole t h r o u g h w h i c h t h e
vitreosil still outlet t u b e projects into the flask. Sterile gauze is w r a p p e d r o u n d the outlet
t u b e above the cover to p r e v e n t d u s t e n t r y a n d to a b s o r b a n y external condensation.
R e c e n t l y m o u l d e d polythene caps h a v e been used.
The stills are cleaned periodically b y immersion in a t a n k of concentrated HNO3
followed b y distilled w a t e r washing. T h e y are t h e n r u n until the c o n d u c t i v i t y of the w a t e r
o u t p u t is satisfactory and the p H w i t h i n n o r m a l rmlge for water.
The m e t h o d s of cleaning used for the 1946 a n d 1948 crucial tests followed closely t h o s e
described in the previous p a p e r s to which reference h a s already been made.
 36 Tt{E :BRITISH HOM(:EOPATHIC JOUR]ffAL

APPENDIX D
ACCESSORY CONTROL PROCEDURES
D1. Bacteriological factors i n starch and diastase. Possible effects of b. subtilis.
Over the years bacteriological tests h a v e been carried o u t b y Dr. J o h n P a t e r s o n ,
D . P . H . , the late Dr. Win. Briggs, Clinical Pathologist, G . H . H . , a n d in 1952 b y Dr. J. C.
I r e s , Bacteriological D e p a r t m e n t , Glasgow R o y a l I n f i r m a r y , and :N. Cram, P h . D . , tech-
nician, G . H . H . , to all of w h o m I a m sincerely grateful. The r e l e v a n t control results were
those obtained in ] 952, t h o u g h earlier filtration e x p e r i m e n t s led up to t h e m .
(a) The distilled water, w h e n carefully handled, w a s sterile on culture, a n d the r o u t i n e
for all t e s t s ensured this. Culture m e t h o d s to cover fungi, spores a n d bacteria were negative.
(b) The soluble s t a r c h was, w i t h m o s t bottles, f o u n d to h a v e an atypical coliform
g r o w t h b u t no b. subtilis. This was found also w i t h the s t a r c h solution even after the
r o u t i n e boiling. Various sterilization procedures, including U.V., were tried b u t gave
a n o m a l o u s results. Sintered glass v a c u u m filtration of a sufficiently fine character could n o t
be used because of the slight viscosity of the solution. Sterilization w a s finally obtained b y
s p r e a d i n g the p o w d e r o u t in a large beaker a n d h e a t i n g in an oven f r o m r o o m t e m p e r a t u r e to
150 ~ C. for 189hours. The solution m a d e thereafter w a s a little m o r e fiocculent and h a d to be
filtered t h r o u g h a sterile B u c h n e r funnel a n d 30 W h a t m a n sterile filter paper. Culture of
this solution was negative. I n tests sterile s t a r c h solution w a s f o u n d to be equally suitable,
the controls falling w i t h i n the l~ormal range of Spekker reading.
A live emulsion of b. subtilis cultured f r o m t h e original diastase w a s then added to a
n o r m a l starch solution w i t h o u t diastase, t h u s giving a far higher bacterial concentration
t h a n normal. Two s i m u l t a n e o u s s a m p l i n g tests, one s t a r c h - d i a s t a s e , the o t h e r starch-
emulsion w i t h o u t diastase, showed no effect f r o m the emulsion over the time of incubation
(Fig. 21). Again, samples of s t a r c h - w a t e r and s t a r c h emulsion gave after n i n e t y m i n u t e s
incubation no Spekker difference b e t w e e n the samples. As one s t a r c h solution was c o m m o n
to all flasks in the crucial tests there w a s t h u s no a d v a n t a g e in sterilization except for
accessory controls.
I-C

z
s
~t

O~
Off
5cai, O ~ ~ 0 0 0-----0

I I I I I I I
60 70 80 90 I00 110 120 130
TIME IN M I N U T E S

FIG. 21
Two s a m p l i n g tests r u n simultaneously. Soluble s t a r c h 5 g. in 500 ml. distilled water.
B . D . H . diastase 0.15 g. in 100 ml. distilled water, filtered W h a t m a n 40. Bacterial emulsion
(5 X 109 per ml.) f r o m culture of b. subtilis f r o m the diastase, 1 ml. in 47 ml. distilled w a t e r
giving 1.04 X l0 s per ml. used in second test in place of 48 ml. diastase solution. Bacillary
c o u n t in n o r m a l diastase solution = 2 x l0 s per ml. 9 F i r s t test n o r m a l starch-diastase
solution; 0, second t e s t starch-emulsion solution. :No m e a s u r a b l e hydrolysis f o u n d w i t h
starch-emulsion w i t h o u t diastase. The quantities used were those suitable for a n i n e t y
m i n u t e incubation w i t h paired flask tests.

(c) Malt diastase w a s u n i f o r m l y f o u n d to yield organisms of b. subtili8 g r o u p on culture.

U.V. sterilized the solution b u t n o t the powder, b u t the activity of the diastase w a s irregu-
larly affected as w a s expected. I t w a s found possible to sterilize t h e solution b y v a c u u m
filtration t h r o u g h sintered glass :No. 5. U n f o r t u n a t e l y , this procedure, t h r o u g h adsorption,
g r e a t l y reduced the a c t i v i t y of t h e reaction w i t h the usual solution, p r o b a b l y due to change
in concentration. B y increasing t h e q u a n t i t y of diastase in the solution before filtration this
could be c o m p e n s a t e d for, b u t no w a y could be f o u n d of r a p i d l y s t a n d a r d i z i n g the r e s u l t a n t
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 37

solution because of the a d s o r p t i o n variation, n o r in practice of keeping the filtration time etc.
w i t h i n the limited period required to p r e v e n t partial inactivation developing before the
d a y ' s run. K e e p i n g the solution longer led to a r a t h e r wider change of control results t e n d i n g
t o w a r d s lower control limits. The s a m p l i n g tests w i t h b. subtilis r e m o v e d in this w a y s h o w e d
the u s u a l general t y p e of curve b u t w i t h small d a y to d a y shifts due to c o n c e n t r a t i o n
differences a n d showed t h a t as in o r d i n a r y s a m p l i n g t e s t s only v e r y small changes of p H
occurred over the i n c u b a t i o n time (Fig. 4). This confirmed t h a t the presence of b. subtilis
h a d no observable effect on the pI-I.
I t was decided, therefore, to find to w h a t extent, if any, b. subtilis influenced the r a t e
of the reaction. An average c o u n t of bacteria in the flasks u n d e r n o r m a l e x p e r i m e n t a l
conditions was made, giving a b o u t t w o million per cc. A live emulsion was m a d e in the s a m e
distilled w a t e r f r o m a n agar slope of the subtilis organisms. Firstly, as already stated, t h e
hydrolytic rate of s t a r c h alone plus emulsion containing a b o u t 5,000 million per cc. w a s
checked against starch hydrolysis alone, a n d no difference observed. Secondly, a diastase
solution filtered t h r o u g h sintered glass a n d freed f r o m organisms, w a s m a d e w i t h Sufficient
e x t r a diastase to give, after filtration, an a p p r o x i m a t e l y usual rate of hydrolysis as judged
b y p r e v i o u s sampling tests. To this diastase solution was added 1 co. of emulsion (5,000
million per co.) m a d e in the same distilled w a t e r as was used for tests. A 16 flask r u n gave
control w i t h i n p e r m i t t e d limits. I t w a s evident, therefore, t h a t even a gross increase in
organisms of the subtilis t y p e usually p r e s e n t did n o t m o d i f y t h e t y p e of control result obtained
in the crucial tests. As the organisms, in m u c h smaller concentration, were c o m m o n to b o t h
control a n d microdose tests the complications p r o d u c e d b y v a c u u m sintered glass steriliza-
tion outweighed a n y a d v a n t a g e a n d these m e t h o d s were, therefore, only retained for
accessory controls. I n a n o r m a l test r u n c o u n t s were m a d e of the organisms a n d a n y average
increase over the incubation time of n i n e t y m i n u t e s w a s p r o p o r t i o n a t e l y negligible, as com-
p a r e d w i t h the high concentration of organisms in the accessory controls described.
I t w a s concluded t h a t the presence of organisms as f o u n d on culture did not, in the con-
centrations f o u n d a n d over the period of the experiment, affect the tests or provide a n y
cause for the stimulative effects obtained.
D2. Variable factors in the starch-iodine complex which might cause change in the A.G. V.
of solutions.
(a) Possible colour change due to t e m p e r a t u r e effects on solutions while in t h e Spekker
cells. Analysis of a v e r y large n u m b e r of sessions showed a m a x i m u m variation of 0 . 2 ~ C.
b e t w e e n the pair of Spekker cells at time of examination. These small t e m p e r a t u r e varia-
tions were unrelated to the results a n d were negligible.
(b) Possible c o n t i n u a t i o n of hydrolysis w i t h change of A.G.V. during time of measure-
m e n t . The Lugol addition slowed hydrolysis considerably. A series of m e a s u r e m e n t s of the
A.G.V. of the same solutions t h i r t y m i n u t e s after the n o r m a l e x a m i n a t i o n time gave an
average change of 0" 025 u p w a r d s . The a m o u n t of change t h a t could take place during t h e
actual time of e x a m i n a t i o n (two minutes) of the p a i r o f Spekker cells for the first m e t h o d was,
therefore, well w i t h i n 0. 005 on the absorptiometer. F u r t h e r , the differences for statistical
p u r p o s e s were those o b t a i n e d b y the first m e t h o d of procedure. The second m e t h o d of
m e a s u r e m e n t carried o u t immediately after acted as a continual check on a n y change of
colour causing a f u r t h e r difference. No additional differences relating to time were found.
The accuracy of the pipetting, the cooling a n d the h e a t protection are dealt w i t h in t h e
text.
D3. Effect of HgCl 2 on starch-iodine colour.
(a) I o d i n e 1:100 Lugol as used for crucial tests. HgC12 in distilled w a t e r .
I
Solution ! Spekker R e a d i n g Against
! Distilled W a t e r at 1.0

1. Iodine : water 1:3 Control .-i-I- 0.89

2. Iodine : HgCI 2 10 -5, 1:3 .. 0.89
3. Iodine : HgCl~ 10 --4, 1:3 ! 0.89 to 0.90
4. Iodine : HgC12 10 -3, 1:3 0.96
5. Iodine : HgC12 10 -2, 1:3 0.99 to 1.0
r

(b) H y d r o l y s i s t e s t s using in place of microdose, HgC12 dilutions in distilled w a t e r as in

usual procedure. Lugol iodine 1:100, 1 ml. added after incubation.

Concentration of HgC13 Colour After Adding Lugol I o d i n e

1. I-IgC13 10 -3, 3 ml. Blue off scale, colourless on shaking. (4 ml. Lugol iodine
1:100 gives p e r m a n e n t blue.)
2. HgC1, 10-4, 3 ml. Blue, off scale, colour r e m a i n e d after shaking.
3. HgCI~ 10 -5, 3 ml. Blue, off scale colour r e m a i n e d after shaking.
38 THE BRITISH HOM(EOPAT}tIC JOURNAL

W i t h high concentration of HgC12 a chemical reaction t a k e s place b e t w e e n HgCI~ and

Lugol iodine affecting the starch-iodine colour. W i t h concentration less t h a n 10 - 4 no effect
w a s f o u n d on the colorimetrie d e t e r m i n a t i o n w i t h Lugol h l 0 0 w i t h quantities used. The
Lugol iodine used w a s a solution of 12 in K I w i t h distilled w a t e r tested spectrographically
for p u r i t y .
D4. Interchange of Glassware. Control exclusion of adsorption f r o m p r i o r molecular
solutions of mercuric chloride as an e x p l a n a t i o n of results.
Two m a i n series were selected as examples.
(a) 1946. W i t h i n the m a i n series giving a high significance 105 tests were r u n where the
second flasks of the control blanks (30 pairs) were given 3 ml. of w a t e r succussed after one
d r o p of distilled w a t e r h a d been added, a procedure exactly the same as t h a t used in prepar-
ing microdoses, b u t w i t h o u t HgC12. The interchange of pipettes, flasks a n d bottles for this
set of tests is given.
Pipettes. Seventeen pipettes chosen at r a n d o m were used on 210 occasions; eleven were
f o u n d to have been used for all solutions including control w a t e r (23 times) a n d microdose
(53 times); seven were used fifty-three times chiefly in the order control-microdose-control-
microdose, while three were used for either control w a t e r or diastase a n d either microdose
or test flask diastase. Two p i p e t t e s were u s e d for test flasks only (five times).
Flasks. T h i r t y - t w o flasks chosen at r a n d o m were used on 210 occasions for all solutions,
including control w a t e r (135 times) a n d microdose (75 times). Ten occurred chiefly in the
order control-microdose-control-microdose, the remaining t w e n t y - t w o all showing alterna-
tion at intervals.
Bottles. AU botties over the years were picked a t r a n d o m for w a t e r or microdose f r o m a
pool of a b o u t n i n e t y bottles w i t h the result t h a t t h e interchange of use for either control or
microdose was general. I n a particular series where n u m b e r s were fewer the r a n d o m m e t h o d
gave three classes, (1) bottles used for b o t h control w a t e r a n d microdose, (2) bottles used
for microdose only in the series, a n d (3) bottles used for control only. I n this 1946 block of
tests nineteen bottles were used at r a n d o m on n i n e t y - t h r e e occasions b o t h for control (thirty-
four times) a n d microdose (fifty-eight times). These l a t t e r included the bottles used in
p r e p a r i n g microdoses. The first flask of b o t h control blanks a n d test pairs received the same
distilled w a t e r f r o m a c o m m o n flask f r o m which the w a t e r for m a k i n g microdoses on t h a t
d a y h a d been taken. I n addition five bottles were used once for control only a n d fourteen
were used once for microdose only in this series, b u t were t a k e n f r o m the c o m m o n pool in
general w o r k for control p u r p o s e s also.
(b) 1952. Special series of "blindfold" tests for unconscious bias w h e r e results were only
defined as control or microdose b y a code after the technician h a d completed the d a y ' s r u n
w i t h o u t knowledge of w h i c h solutions were being used. The glassware exchange is given for
the $31 a n d $27 (ordinary HgC12) tests.
Pipettes. Sixteen p i p e t t e s were used 218 times for w a t e r a n d diastase, b u t were k e p t
s t r i c t l y in two classes, eight for control w a t e r (fifty-four times) or microdose (fifty-five times)
a n d the other eight for diastase control flasks (fifty-ibur times ) a n d test flasks (fifty-five
times). All w a t e r a n d diastase p i p e t t e s occurred w i t h at least t w o alternations at intervals of
control a n d microdese.
Flasks. T w e n t y flasks were picked at r a n d o m for all solutions a n d used 218 times.
E i g h t e e n of these were used for control flasks (154 times), a n d for test flasks (fifty-one
times). F o u r pairs of flasks were used once for control or tests, unlisted. One flask w a s used
once for microdose only, while one flask w a s used four times for control only. Ten flasks
occurred chiefly in the order of control-microdose-control-microdose, while eight others were
f o u n d to h a v e been alternated at intervals.
Bottles. F i f t y - f o u r bottles chosen at r a n d o m f r o m the c o m m o n pool w e r e used on 107
occasions, of these t w e n t y bottles were f o u n d to h a v e been used on fifty-eight occasions
b o t h for microdose ( t h i r t y tinges) a n d control (twenty-eight t i m e s ) - - s o m e for control before
microdose and others for microdose before control, a n d some for microdose before a n d after
control. 1Yineteen of these microdose readings were over 0' 30. Seventeen bottles were used
for microdose only on t w e n t y - f o u r occasions giving eight readings over 0" 30. Seventeen
b o t t l e s were used for control only on twenty-five occasions, while of these latter control
b o t t l e s it was f o u n d t h a t ten h a d been used prior to this series for microdose.
These m e t h o d s of using glassware at different times b o t h for control a n d microdose
solutions were e m p l o y e d in all the 1952 series. T h e y a p p e a r to exclude a n y " e x p l a n a t i o n "
o f t h e " h i g h p o t e n c y " microdose action as being due to molecules of HgCl~ adsorbed by
chance on glassware on some r e m o t e previous occasion w h e n molecular doses were being
tested, especially w h e n the cleaning m e t h o d s are also t a k e n into consideration.
D5. Possible contamination of prepared mierodoses.
(a) A set of separate $27 microdoses were m a d e f r o m $26, to ensure correct
s t a g e s of p r e p a r a t i o n . T h e y were t h e n added t o g e t h e r to provide sufficient solution
for spectrographic examination. A 16-flask t e s t w a s t h e n r u n using this larger $27 supply-for
~he microdoses. Marked stimulative results w e r e o b t a i n e d along w i t h n o r m a l controls. The
s u p p l y flask was t h e n sent u n t o u c h e d to Messrs. J o h n s o n M a t t h e y along w i t h a control flask
o f the same s a m p l e of distilled w a t e r f r o m which t h e y h a d been mad6.
W i t h i n the range of elements t e s t e d for a n d the limits of the spectrographic anaiysis,
a b o u t 0- 1 P.Prm. for Hg, the microdose solution giving stimulative results showe d n o ex?Frm!-
 EVIDENCE OF THE ACTIVITY OF HIGH POTENCIES 39

eous c o n t a m i n a n t s . I t is to be n o t e d t h a t , as e x p e c t e d , no H g w a s f o u n d p r e s e n t i n t h e mi c ro-
dose (Table X).
TABLE X
SPECTROGRAPHIC ANALYSIS OF SAMPLES OF DISTILLED WATER AND IV[ICRODOSES IN THE
S A M E DISTILLED W A T E R . T H E V A L U E S A R E IN PARTS P E R M I L L I O N (P.P.M.).

Flasks D i s t i l l e d W a t e r (Control) Microdose i n D i s t i l l e d W a t e r

No. 101 2Vo. 112 1go. 122 No. 134

Calcium .. 0.5 0.5 0.4 0.5
Magnesium .. 0-2 0-2 0.2 0.2
Silicon . . . . 0.2 0.4 0.2 0.2
Iron . . . . 0 . 1 to 0-2 0.05to 0.1 0.1 0.1
Copper . . . . 0.1 0.1 0.1 [
i 0.05
S o d i u m .. 0 . 1 to 0-2 0 . 1 to 0 . 2 0.1 to 0.2 0.1 to 0.2
Alumininm (if a n y ) 0.05 0-05 0.05 ! 0-05
Boron* . . . . <2 <2 <2 <2

* I t is difficult to e s t i m a t e t h e a m o u n t s as B o r o n is p r e s e n t i n t h e g r a p h i t e electrodes.
T h e followin g e l e m e n t s were n o t d e t e c t e d in a n y sa mpl e s : Ag, B a , Bi, Cd, Co, Cr, H g ,
K, Li, Mn, Ni, Pb , Sb, Sn, Zn.
I t w o u l d a p p e a r f r o m t h e a b o v e r e s u l t s t h a t t h e r e is no s i g n i f i c a n t difference in com-
p o s i t i o n b e t w e e n t h e s a m p l e s in t h e t w o cases.

E x a m i n a t i o n of a s i m i l a r $27 m i c r o d o s e w i t h t h e e l e c t r o n m i c r o s c o p e w a s c a r r i e d o u t
b y Mr. J. W. S h a r p a t t h e R o y a l T e c h n i c a l College, to w h o m I a m m o s t g r a t e f u l . N o s i gn of
a n y v i r u s - l i k e bodies w a s found, w h i l e t i l e electroi~ d i f f r a c t i o n p a t t e r n of t h e c r y s t a l l i n e
r e s i d u e of t h e m i c r o d o s e w a s e s s e n t i a l l y s i m i l a r to t h a t of t h e c o n t r o l d i s t i l l e d w a t e r .
(b) T h e d i s t i l l e d w a t e r p r e p a r e d a n d u s e d as r o u t i n e w a s s u b m i t t e d for a t e n t a t i v e fl a me
s p e c t r o p h o t o m e t e r test. This w a s m u c h less s e n s i t i v e t h a n t h e s p e c t r o g r a p h i c a n a l y s i s , o n l y
N a b e i n g d e t e c t e d . E x p e r t a d v i c e w a s s o u g h t , in m a n y q u a r t e r s , r e g a r d i n g v a r i o u s p h y s i c a l
m e t h o d s of a n a l y s i s , b u t i t w a s clear t h a t for t h e l i m i t s r e q u i r e d t h e s p e c t r o g r a p h w a s t h e
b e s t a v a i l a b l e m e a n s for d e t e c t i o n of e l e m e n t s in m i n u t e t ra c e s . The b a c t e r i o l o g i c a l e x a m i n -
tion, as s t a t e d p r e v i o u s l y , w a s n e g a t i v e . T h e r e r e m a i n e d pos s i bl e ga s v a r i a n t s t h a t m i g h t
o c c u r d e r i v e d f r o m o r i g i n a l t a p w a t e r , l a b o r a t o r y air or b u n s e n b u r n e r s . N o b u n s e n s w e re
p e r m i t t e d in t h e a c t u a l e x p e r i m e n t a l l a b o r a t o r y a n d a l a r g e e x t r a c t o r f a n w a s k e p t r u n n i n g
n e a r t h e d o u b l e stills a n d b o i l i n g a p p a r a t u s in t h e s t i l l room. T h e r o u t i n e d o u b l e d i s t i l l e d
w a t e r f r o m a p y r e x flask w a s a n a l y s e d b y Messrs. T a t l o c k a n d T h o m s o n , P u b l i c A n a l y s t s .
T h e r e s u l t is s h o w n in T a b l e X1. The r e s u l t s i n d i c a t e d t h a t t h e t r a c e s f o u n d w e re t h o s e
n o r m a l l y p r e s e n t in a c i t y l a b o r a t o r y a t m o s p h e r e a n d w o u l d be c o m m o n t o a l l s ol ut i ons .
CO 2 w a s t e s t e d for in d i s t i l l e d w a t e r e x p o s e d in a n open d i s h i n t h e l a b o r a t o r y d u r i n g s e v e r a l
w o r k i n g h o u r s on different occasions in order to g e t t h e m a x i m u m a b s o r p t i o n .

TABLE XI

CHEMICAL ANALYSIS OF DISTILLED WATER

P a r t s P e r Million

Carbon Dioxide* . . . . 2-4

Sulphur Dioxide .. <0"6
S u l p h u r i e A c i d (Sulpl~i'des) .. <0.5
N i t r i c A c i d (Nitrites) .. <0"3
N i t r o u s A c i d (Nitrites) .. 9 <0-1
Ammonia . . . . . . * 9 none
Chlorides . . . . . . . , none
Phenols . . . . . . 9 none

* D i s t i l l e d w a t e r p r e v i o u s l y e x p o s e d in o p e n dish in l a b o r a t o r y for s e v e r a l hours.

The p o s s i b l e effect of CO 2 is d i s c u s s e d in A p p e n d i x D6. T h e t e s t s d e s c r i b e d u s i n g

d i s t i l l e d w a t e r p r e v i o u s l y s u c c u s s e d also m e e t s a n y s u g g e s t i o n t h a t t h e s t i m u l a t i v e r e s u l t s of
m i c r o d o s e s m i g h t in s o m e w a y be d u e solely to t h e s u c c u s s i o n of t h e w a t e r u s e d in t h e
p r e p a r a t i o n of t h e doses.
D6. Effect of CO 2 Concentration.
A l t h o u g h t h e p l a n n i f l g of t h e e x p e r i m e n t p r o v i d e d for i n t e r c h a n g e of p o s i t i o n of
c o n t r o I a n d t e s t flasks in t h e i n c u b a t i n g b a t h f r o m d a y t o d a y , g i v i n g e x a c t l y t h e s a m e
c o n d i t i o n s o v e r a p e r i o d of t e s t s for all flasks, t h e s u g g e s t i o n h a s b e e n m a d e t h a t a n a p p a r e n t
40 THE BRITISH HOM(EOPATHIC JOURNAL

s t i m u l a t i o n m i g h t be due to the first flask of a pair of test flasks being p a r t l y deactivated

either b y concentrations of CO s in t h e a t m o s p h e r e above the b a t h , or b y the b r e a t h of t h e
technician. As the first flask is used as a s t a n d a r d in the first m e t h o d of procedure, this
w o u l d m a k e the Spekker reading of the second flask a p p e a r relatively higher as if stimula-
tion h a d t a k e n place. The chance effect of localized concentrations w a s excluded b y the
daily interchange of the position of the test a n d control flasks in t h e b a t h a n d b y statistical
assessment. The effect of b r e a t h w a s checked b y t h e technician b r e a t h i n g directly into
selected flasks in a 16 flask test w i t h no sign of a n y effect against controls. E x p e r i m e n t a l l y ,
b r e a t h i n g into flasks only increased c o n d u c t i v i t y b y a b o u t 0" 1 [xmho. Assuming, however,
an occasional chance effect, while this would escape notice in the first procedure where t h e
first flask was set a t 0.30, the second m e t h o d used t h r o u g h o u t in 1952 which c o m p a r e d all
t h e flasks against distilled w a t e r at 1.0 would h a v e excluded such an a b e r r a n t flask. T h u s it
was d e m o n s t r a t e d t h a t the second flask readings were reliable w h e n showing a s t i m u l a t i v e
effect.
If, on the o t h e r h a n d , a chance effect of e x t r a C02 were to h a v e altered the p H , t h e n in
order to affect definitely the result it would h a v e required to be capable of p r o d u c i n g in the
flasks a wide change sufficient to give a p H outside the range 5 . 0 to 5-5 (see section on
Buffers), a m u c h larger effect t h a n was likely u n d e r the e x p e r i m e n t a l conditions, a n d one
easily checked b y c o n d u c t i v i t y m e a s u r e m e n t s . If, on the o t h e r hand, t h e v e r y d o u b t f u l
a s s u m p t i o n is m a d e t h a t v a r y i n g small traces of CO s in the second flask m i g h t give a
chemical stimulation w i t h o u t perceptible p H change it is to be r e m e m b e r e d t h a t there
would already be a certain v a r y i n g m i n i m u m of CO s in b o t h t e s t a n d control flasks t h r o u g h -
o u t the series which should, on this a s s u m p t i o n , h a v e p r o d u c e d r a n d o m s t i m u l a t i o n in
controls also. This did n o t occur.
Again, the distilled w a t e r used for original solutions w a s c o m m o n to all flasks a n d the
exposure of beakers to the a t m o s p h e r e was c o m m o n to the original solutions. The only set
variables were the succussed microdose solutions, a n y other c o m m o n chance variables being
t a k e n care of b y the statistical analysis. E v e n if we m a k e the v e r y d o u b t f u l a s s u m p t i o n t h a t
e x t r a COs in the microdoses m i g h t h a v e caused stimulation, c o n d u c t i v i t y tests w i t h succus-
sion of w a t e r only showed a rise of a b o u t 1 ~zmho for the ten suceussions, the bottles being
stoppered. This was negligible, and no m o r e t h a n m i g h t be found as a variation in a control
bottle. N o t only so, b u t a g r o u p of controls, p a r t of one series of crucial tests giving high
significance, w a s carried out using, for the second flask of t h i r t y pairs of control blanks,
water f r o m similar bottles also succussed after t r a n s f e r of one drop of distilled w a t e r f r o m a
prior bottle, exactly as in microdose preparation, b u t w i t h o u t , of course, the original
mercuric chloride for microdose. The test flasks received microdoses p r e p a r e d w i t h succus-
sion in the s t a n d a r d way. No control a b n o r m a l i t y was found.
I n v e s t i g a t i o n s were carried o u t to find w h a t concentrations of CO s in w a t e r used in
place of microdose solutions could partially deactivate the hydrolysis, using s a m p l i n g a n d
16 flask tests. I n the latter a concentration of over 60 p . p . m , w a s required to s h o w a n y
such effect, an a m o u n t m u c h greater t h a n a n y possible chance e x p e r i m e n t a l variation.
F u r t h e r , in a n o t h e r n o r m a l 16 flask test, the average sp. c o n d u c t i v i t y difference b e t w e e n
the two m e m b e r s of a n y pair of flasks after i n c u b a t i o n b u t before Lugol was added w a s
less t h a n 0"5 ~zmhos a t 36 ~ C., while no relationship w a s f o u n d b e t w e e n the small conduc-
t i v i t y differences a n d the A.G.V. readings of different flasks, indicating no differences
sufficiently large to indicate u n u s u a l increase of COs arising b y chance in a n y single flask.
Again, t a k i n g the distilled w a t e r of the quality used (see Tables I I , X, a n d X I ) w i t h a
sp. c o n d u c t i v i t y u n d e r 1 ~zmho, it w a s a s s u m e d t h a t a n y additional rise in c o n d u c t i v i t y
w i t h i n the period of an e x p e r i m e n t a l session was p r e d o m i n a n t l y due to COs absorption.
After s t a n d i n g for a s h o r t t i m e t h e sp. c o n d u c t i v i t y of the w a t e r if in stoppered flasks
r e m a i n e d at a p p r o x i m a t e l y 1.5 ~tmhos. A useful a p p r o x i m a t i o n of CO s a b s o r p t i o n relative to
conductivity of the w a t e r at 17 ~ C. w a s obtained w i t h dilutions of a solution of COs, of w h i c h
the concentration in p . p . m , was f o u n d b y analysis. Table X I I gives the values arrived at.

TABLE X l I

Sp. Conductivity COs in solution

at 17 ~ C. in ~ m h o s in p . p . m .

10"0 100
6"9 50
5"2 25
4'0 13
2-8 6
2"2 3
2'0 1-2

Sp. c o n d u c t i v i t y 0f distilled w a t e r at 17 ~ C. relative to p . p . m , of CO s insolution= Approxi-

mate values u n d e r the experimental conditions described.
 :EVID]~I~CE OF THE ACTIVITY OF HIGH POTENCIES 4]

T h e r e w a s t h e n a d d e d to a series of flasks, c o n t a i n i n g t h e s t a n d a r d q u a n t i t y of s t a r c h
d i a s t a s e solution, 3 ml. of distilled w a t e r w i t h t h e c o n d u c t i v i t y v a l u e altered b y a d d i t i o n elv
CO2, t h e p H of e a c h flask b e i n g m e a s u r e d . I t w a s f o u n d t h a t to p r o d u c e c h a n g e s of a b o u t
0 . 2 p H in t h e s t a r c h - d i a s t a s e solution t h e 3 m l . of w a t e r r e q u i r e d to h a v e a sp. c o n d u c t i v i t y
of a p p r o x i m a t e l y 7 . 6 ~zmhos. A s c a n be seen in Fig. 3 a c h a n g e of 0 . 2 p H could affect t h e
r a t e of h y d r o l y s i s o n l y if t h e p H o f t h e s t a r c h - d i a s t a s e w a s a t t h e lower limit of t h e s t a b l e
region b e t w e e n p H 5 . 0 a n d 5.5. A s p . c o n d u c t i v i t y of 7 . 6 ~ m h o s of t h e w a t e r c o r r e s p o n d e d
to 60 p p m . of CO2, a c o n c e n t r a t i o n less t h a n t h a t a l r e a d y f o u n d to affect h y d r o l y s i s . T h e
3 ml. o f w a t e r or m i c r o d o s e a d d e d to e a c h flask could, therefore, o n l y h a v e affected t h e
e n z y m e process if t h e sp. c o n d u c t i v i t y h a d b e e n definitely g r e a t e r t h a n t h a t f o u n d on a n y
occasion. T h u s a n y s m a l l c o n d u c t i v i t y c h a n g e s d u e to CO2 in t h e control or m i c r o d o s e
solutions, a n d f o u n d u n d e r n o r m a l t e s t e x p e r i m e n t a l c o n d i t i o n s were negligible.
I t w a s clear, therefore, t h a t t h e CO 2 c o n c e n t r a t i o n occurring u n d e r t h e c o n d i t i o n s of t h e
e x p e r i m e n t s could n o t a c c o u n t for t h e effects o b t a i n e d . T h e s e c o m m e n t s also a p p l y to t h e
m i n u t e c o n c e n t r a t i o n s of o t h e r g a s e s d e r i v e d f r o m t h e a t m o s p h e r e , a n d f o u n d in t h e dis-
tilled w a t e r c o m m o n to all solutions ( A p p e n d i x D5).
D7. Use of Lissapol N for Cleaning.
Lissapol N is a n a q u e o u s solution of a n a l k y l a t e d p h e n o l e t h y l e n e oxide c o n d e n s a t e ,
t h a t is a s u r f a c e - a c t i v e a g e n t c o n t a i n i n g a p o l y e t h y l e n e glycol chain. I t is a non-ionic
s u r f a c e - a g e n t a n d does n o t ionize in w a t e r a n d does n o t f o r m salts. I t is m o r e soluble in
cold t h a n in h o t water, a n d is r e a d i l y soluble in t h e former.
To d e t e r m i n e t h e possible effect of Lissapol on t h e h y d r o l y s i s of s t a r c h w i t h d i a s t a s e , a
s h o r t series of h y d r o l y s i s t e s t s were r u n u s i n g Lissapol solutions in place of microdoses.
(a) Six pairs of flasks w i t h t h r e e p a i r s of control b l a n k s were t e s t e d u s i n g dilutions o f
p u r e Lissapol in distilled w a t e r r a n g i n g f r o m 1 in 5 to 1 in I00. All t e s t s g a v e a Spekker read-
ing off scale a g a i n s t 0- 30 e x c e p t t h e 1 in 100, w h i c h g a v e a n A.G.V. o f 0" 19 a g a i n s t 0" 30.
P u r e Lissapol in dilutions u p to 1 in 100 therefore i n h i b i t e d h y d r o l y s i s .
(b) T h e first s t a g e of cleaning u s e d a p y r e x t a n k of Lissapol in distilled w a t e r (3 table-
s p o o n s to gallon), i.e. a p p r o x i m a t e l y 1 to 100. A 16 flask t e s t using, for test, solution f r o m
t h e first s t a g e t a n k s h o w e d m a r k e d i n h i b i t i o n o f h y d r o l y s i s .
(e) A 16 flask t e s t u s i n g for t e s t 3 ml. distilled w a t e r f r o m flasks t a k e n f r o m t h e vitreosil
t r o u g h after t h e boiling s t a g e of cleaning g a v e a clear r u n of v e r y e x a c t control r e a d i n g s
=t=0" 01 a b o u t 0-30 i n d i c a t i n g c o m p l e t e f r e e d o m f r o m a n y e x t r a n e o u s c o n t a m i n a n t .
I t is to be n o t e d t h a t following t h e boiling s t a g e t h e r e is, in r o u t i n e cleaning o f flasks
a n d s m a l l bottles, a f u r t h e r s t a g e o f s h a k i n g a n d r i n s i n g w i t h distilled w a t e r followed b y
d r y h e a t i n g a t 150 ~ C. T h e m e t h o d of cleaning w i t h two s t a g e s of s h a k i n g a n d r i n s i n g w i t h
distilled w a t e r followed b y a d e q u a t e boiling, f u r t h e r w a s h i n g a n d d r y h e a t i n g is, therefore,
e n t i r e l y s u i t a b l e a n d in practice e n a b l e s t h e v e r y s a t i s f a c t o r y cleaning a c t i o n of Lissapol to
be u s e d , w i t h exclusion of a n y s u b s e q u e n t effect on hydrolysis.

APPENDIX E
DIFFERENCES OF TECHNIQUE BETWEEN 1946, 1948 A N D 1952
(a) Cleaning
1946, 1948. C o n c e n t r a t e d H N O 3 followed b y s t a g e s similar to t h o s e described after t a n k
s t a g e in A p p e n d i x C.
1952. Lissapol (3 t a b l e s p o o n s to gallon) w i t h p r o c e d u r e as described in A p p e n d i x C.
(b) Pipetting
1946, 1948. P i p e t t e s r u n free, 1946 m o u t h p i p e t t i n g . 1948 s u c t i o n s e m i - a u t o m a t i c p i p e t t i n g .
1952. Pipettes touching surface after running out--suction semi-automatic pipetting.
B o t h m e t h o d s g a v e a p p r o x i m a t e l y t h e s a m e a c c u r a c y as b e t w e e n pipettes.
T h e o u t p u t in 1946, 1948, w a s fractionally less, b u t c o m m o n to all experi-
ments.
(c) Control water, in second flask o f c o n t r o l b l a n k s . T h e s a m e w a t e r a l w a y s u s e d for c o n t r o l s
as for p r e p a r i n g microdoses.
1946. P a r t l y f r o m c o m m o n flask, p a r t l y u s i n g s u c c u s s e d w a t e r f r o m s e p a r a t e b o t t l e s .
1948. F r o m c o m m o n flask.
1952. F r o m s e p a r a t e bottles.
(d) Ab~orptiometer
1946. Old type.
1948. New type.
1952. :New type.
(e) Procedure
1946, 1948. F i r s t m e t h o d only.
1952. B o t h first a n d second m e t h o d s u s e d in all t e s t s in all series.
(f) Diastase
1946, 1948. T h e t i m e o f d i a s t a s e in solution before w a r m i n g in i n c u b a t i o n b a t h n o t s t r i c t l y
laid down.
 42 TIlE BI~ITISII I-IOI~I(:EOPATIIlC JOURNAL

1952. T h e t i m e of m a k i n g d i a s t a s e solution fixed. Solution into i n c u b a t i o n b a t h

immediately.
1946, 1948. D i a s t a s e solution in single b o t t l e for s u p p l y in i n c u b a t i n g b a t h .
1952. D i a s t a s e in s e p a r a t e s m a l l b o t t l e s in i n c u b a t i n g b a t h for s u p p l y for each pair.
This permitted the use of the pipetting method where the pipettes touched
t h e s u r f a c e of flask solution.

APPENDIX F
NOTES ON MICRODOSES
A. Dilution and Homogeneity. T h e order o f d i l u t i o n g i v e n in t h e t e x t is a p p r o x i m a t e
9only, as t h e s t a g e s of dilution in p r a c t i c e were a b o u t 1 in 200, t a k i n g 5 ml. ~ 200 capillary
drops, one capillary drop = 0. 025 ml., t h i s figure b e i n g a n a v e r a g e of a s a m p l e of d r o p s
f r o m f o r t y s e p a r a t e capillary t u b e s . A s a n order of 10 -61 as a t e r m of a c t u a l d i l u t i o n is
m e a n i n g l e s s t h e m i c r o d o s e s are g i v e n a n o m e n c l a t u r e i n d i c a t i n g t h e n u m b e r o f s t a g e s of
:serial dilution w h e r e a t e a c h s t a g e one capillary drop of t h e p r e v i o u s s t a g e is a d d e d to
5 ml. o f distilled w a t e r in a w e i g h i n g b o t t l e a n d t h e b o t t l e is t h e n s u b j e c t e d to m e c h a n i c a l
:shock (succussion). T h u s $28 i n d i c a t e s t w e n t y - e i g h t s t a g e s of dilution. T h e p r i m a r y solution
u s e d is 1 in 100 b y w e i g h t o f m e r c u r i c chloride in w a t e r (S 1). T h e $28 solution is e q u i v a l e n t ,
o n t h e c e n t e s i m a l scale u s e d in h o m c e o t h e r a p y , to w h a t is k n o w n as 32c.
T h e q u e s t i o n as to w h e t h e r t h e p r e p a r a t i o n of m i c r o d o s e s b y s t a g e dilution m i g h t , d u e
to lack of h o m o g e n e i t y , h a v e led to t r a n s f e r of h i g h e r c o n c e n t r a t i o n t h a n w a s e s t i m a t e d ,
r e q u i r e d to be i n v e s t i g a t e d . V a r i o u s theories h a v e b e e n p u t f o r w a r d to s u g g e s t t h i s is so,
q u i t e a p a r t f r o m a d s o r p t i o n , w h i c h is e l i m i n a t e d for t h e i m m e d i a t e p r e p a r a t i o n b y u s i n g
s e p a r a t e bottles. T h e s u g g e s t i o n t h a t a d s o r p t i o n c a u s e s prior c o n t a m i n a t i o n affecting t h e
later u s e of bottles, etc., is dealt w i t h elsewhere ( A p p e n d i x D).
T h e a d e q u a t e h o m o g e n e i t y o f small a m o u n t s of solution d i l u t e d a n d g i v e n m e c h a n i c a l
s h o c k w a s t e s t e d b y e s t i m a t i o n of t h e a m o u n t of m e r c u r i c chloride in successive s t a g e s
w i t h i n t h e m o l e c u l a r r a n g e , w h e r e n o r m a l m e t h o d s of a n a l y s i s were available.
Chemical e x a m i n a t i o n o f t h e m e r c u r i c chloride p o w d e r u s e d for t h e p r i m a r y solution
g a v e H g e q u i v a l e n t to over 99 p e r cent. m e r c u r i c chloride. U s i n g o r d i n a r y chemical a n a l y s i s
~he p r i m a r y S1 s t a g e a n d t h e $2 s t a g e g a v e a q u a n t i t a t i v e e s t i m a t e of t h e e x p e c t e d a m o u n t .
S p e c t r o g r a p h i c e x a m i n a t i o n g a v e clear evidence of H g a t t h e $2 stage, while t h e $4 stage,
of t h e order of 10 - s , w a s b e y o n d t h e limit of detection. U s i n g t h e Feigl s p o t t e s t , dilutions
<)f l : l , 0 0 0 , 1:10,000, a n d 1:100,000 g a v e colour evidence o f s a t i s f a c t o r y i n c r e a s i n g dilution
while t h e $2 s t a g e ~ 1:20,000 fitted in well in t h e series w i t h 11o s u g g e s t i o n of t r a n s f e r of
u n d u e c o n c e n t r a t i o n . (Feigl, 1943) ( A p p e n d i x G).
I t w a s clear t h a t t h e m e t h o d o f dilution g a v e sufficient h o m o g e n e i t y to e n s u r e a n
i n c r e a s i n g dilution w i t h o u t t r a n s f e r of u n d u e c o n c e n t r a t i o n s . T h e c a l c u l a t i o n o f dilution
w i t h i n t h e m o l e c u l a r r a n g e c a n be t a k e n as a p p r o x i m a t e l y correct, a n d t h e a s s u m p t i o n t h a t
n o m o l e c u l a r traces r e m a i n a f t e r t h e S11 s t a g e c a n be accepted.
B. Technique. I t w a s fe]t t h a t it w a s o n l y reasonable, for a n y w o r k e r t e s t i n g t h e
q u e s t i o n o f " h l g h p o t e n c y " microdoses, to a d o p t for t h e p u r p o s e s o f t h e e x p e r i m e n t s c e r t a i n
a s s u m p t i o n s r e g a r d i n g t h e h a n d l i n g o f s u c h microdoses. T h e s e h a v e b e e n t e n t a t i v e l y
a d o p t e d b y m a n y of t h e p h y s i c i a n s p r a c t i s i n g h o m c e o t h e r a p y . T h e a n a l o g y , w h i c h is s u g g e s t e d
o f t r e a t i n g m i c r o d o s e s as bacterial e m u l s i o n s p r o v i d e s a simple m e a n s of o b t a i n i n g t h e
n e c e s s a r y a t t i t u d e o f m i n d to m e e t t h e e x p e r i m e n t a l s i t u a t i o n . T h e a s s u m p t i o n s , as far as
c o n c e r n s t h e s e e x p e r i m e n t s , are:
1. Microdoses c o n t a i n a n active " p r o p e r t y " w h i c h c a n be affected or h a v e its specificity
a l t e r e d b y c o n t a c t w i t h a similar " p r o p e r t y " in a n o t h e r m i c r o d o s e of different p r i m a r y
origin. I n t h e p r e p a r a t i o n of m i c r o d o s e s t h e d i l u e n t s h o u l d be t h e s a m e distilled w a t e r for all
:stages.
2. Microdoses keep t h e i r a c t i v i t y for long periods if k e p t cool, p r o t e c t e d f r o m d u s t , a n d
n o t e x p o s e d for long periods to s u n l i g h t or U.V.
3. I t is difficult to g e t rid of m i c r o d o s e a c t i v i t y f r o m glassware or a b s o r b e n t surfaces u n -
less b y d r y h e a t , p r o d u c i n g n e a r chemical d r y n e s s . R e c e n t biological e x p e r i m e n t s h a v e sup-
ported this assumption.
4. Microdoses in solution m a y a c t i v a t e a t s h o r t distances, s a m p l e s o f w a t e r , a n d t h e
" a c t i v i t y " m a y also be t r a n s f e r r e d b y c r o s s - c o n t a m i n a t i o n as w i t h b a c t e r i a l e m u l s i o n s .
H e n c e a n y solution spilt o n b e n c h e s or elsewhere m u s t be carefully dried a n d e v a p o r a t e d b y
h e a t w i t h a d e q u a t e v e n t i l a t i o n . R e c e n t biological e x p e r i m e n t s u s i n g " h i g h p o t e n c y " micro-
d o s e s distilled a f t e r b e i n g m a d e h a v e s u p p o r t e d t h i s a s s u m p t i o n .
5. H a n d s c o n t a m i n a t e d b y m i c r o d o s e m u s t be t h o r o u g h l y w a s h e d w i t h soap a n d
running water.
I f s u c h a s s u m p t i o n s are t e m p o r a r i l y a c c e p t e d it will be clear, for e x a m p l e , t h a t one
s h o u l d n o t use a p i p e t t e for a m i c r o d o s e first a n d t h e n for a control w a t e r s a m p l e a f t e r simple
rinsing. C o n d u c t i v i t y cells m u s t be sterilized b y d r y h e a t after b e i n g d i p p e d into microdose,
u n l e s s k e p t for one k i n d o f m i c r o d o s e only. T h i s i n v o l v e s f r e q u e n t c h e c k i n g of cell c o n s t a n t s .
All m i c r o d o s e b o t t l e s m u s t be k e p t s t o p p e r e d a n d , w h e n open, k e p t a p a r t f r o m open w a t e r
c o n t r o l s etc. T h e y are b e t t e r to be k e p t covered b y glass covers tO p r o t e c t t h e m a g a i n s t
(lust a n d U.V. r a d i a t i o n . All g l a s s w a r e u s e d for r i n s i n g m u s t be correctly cleaned a n d
 EVIDENCE OF THE ACTIVITY OF HIGH POTEI~(31ES 43

h e a t e d before being used for f u r t h e r control or test experiments. The acceptance of these
9a s s u m p t i o n s for the e x p e r i m e n t s will e x p l a i n m u c h of the detail given in the t e x t of the
paper.
The a d o p t i o n of these a s s u m p t i o n s was, b r o a d l y speaking, f o u n d in the early years of
this research to i m p r o v e the accuracy of controls, a n d to lessen the occurrence of a b e r r a n t
controls. As m o r e precautions, based on the a s s u m p t i o n s , were introduced, the m o r e
reliable b e c a m e the results. While this in no w a y s u b s t a n t i a t e d the actual a s s u m p t i o n s ,
t h e use of t h e m was felt to be justified, as t h e y supplied a w o r k i n g h y p o t h e s i s t h a t led to
g r e a t e r c o n s t a n c y of results. I t is, therefore, r e c o m m e n d e d t h a t in a n y repetition of these
.experiments this course should be followed.

AI~PENI)IX G
RADIO-ISOTOPE EXPERIMENTS
Mercury ~~ has a gamma energy of 0.28 ~leV and a beta energy of 0" 2] MeV. The
, s~nsitivity of a Scintillation Counter with a sodium iodide phosphor was compared with a
Geiger Counter and it was found that at best working conditions it gave a larger ratio of
~ource to background than the Geiger Counter. Standard dilutions of Hg~oaCl2 were
prepared and comlted in the Scintillation Counter and a graph drawn to show ratio of
concentration to counts per minute (Fig. 22).
,o~M

,o~ -

o
o

,o I _~ I I I I I
Jo-' *o-s to'" Jo"6 tO" ,o'" ,o"

Fro. 22
~ r a p h showing ratio of concentration to counts per minute, using standard dilutions of
Hg2~ 13.
Homogeneity
Dilutions were t h e n p r e p a r e d b y serial dilution in s e p a r a t e bottles as described in t e x t
(see Reagents), w i t h shaking, the succussion a p p a r a t u s n o t being available. The radio-
a c t i v i t y of the stages of dilution was c o u n t e d a n d f r o m the g r a p h of the results it w a s s h o w n
t h a t t h e c o n c e n t r a t i o n fell b y 100 to 1,000 at each stage, t h u s showing t h a t the solutions
w e r e a p p r o x i m a t e l y homogeneous.

Adsorption
An e x t r e m e l y active solution whose c o u n t i n g r a t e was too fast to c o u n t w i t h the
d e k a t r o n , w a s left s t a n d i n g in a bottle for t w e n t y - f o u r hours. The p H of the solution was
a d j u s t e d to 5' 8. I t was rinsed o u t a n d the bottle cleaned as described in cleaning procedure
(Appendix C). The cleaning m e t h o d w a s p r o b a b l y n o t as rigorous as n o r m a l owing to lack
o f the u s u a l e q u i p m e n t . Distilled w a t e r w a s aUowed to s t a n d in the cleaned bottle for
t w e n t y - f o u r h o u r s - - s h a k i n g a t intervals. This w a t e r w a s p u t into a control bottle a n d w a s
t h e n tested a n d f o u n d to have a c o u n t i n g r a t e the s a m e as the b a c k g r o u n d c o u n t indicating
no evidence of c o n t a m i n a t i o n of the distilled w a t e r f r o m a d s o r b e d Hg~0aC12. P a r t of the
.cleaned bottle w a s t h e n g r o u n d d o w n a n d f o u n d to have adsorbed a p p r o x i m a t e l y 6 x 10 -8.
ttgt~ f r o m a solution containing 1-25 X 10 -~ g. of the isotope, i.e. 5 ml. of 1-400
Hg~83C12. This m u s t h a v e been v e r y firmly adsorbed since it did n o t come off w h e n the w a t e r
~vas allowed to s t a n d for t w e n t y - f o u r h o u r s i n t h e bottle as described. I t would seem, therefore,
44 THE BRITISH HOM(EOPATHIC JOURI~!AL

t h a t the thorough cleaning m e t h o d s used in the technique were sufficient to remove a n y

HgCI~ which h a d been less firmly adsorbed. Leeser a n d J a n n e r (1953) with phosphoric acid
solutions containing radio-active phosphorus (p3~) in a single bottle, used for several
solutions, found a detectable a m o u n t given off into the s u b s e q u e n t dilution when only simple~
dilution was used w i t h o u t rigorous cleaning m e t h o d s between stages.

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