CsH, which
is an analogue of murexide.
It is probable that similar changes take place in the case of quinone and
amino-acids. Starting from arbutin, where the hydroquinone which is formed
is oxidised in part to quinone, it is the ammonia from the amino-acid which
is the essential factor in producing the red colour. The aldehyde derived
from the amino-acid plays only an accessory part, for the reduced quinone is
already present. In fact, the red is instantly produced when ammonia is
added to the oxidised mixture. The addition of formaldehyde to hydrolysed
and oxidised arbutin has no effect in producing colour, and this is in agree-
The Formation of the Anthocyan Pigments of Plants. 128
ment with the fact that the red colour is produced no matter what
amino-acid be used. A series of similar experiments was made differing
only in that different amino-acids in equivalent quantity were added in the
several cases. The colours produced were compared with one another by
means of the tintometer in order to obtain a rough indication of their
relations with one another. The same series was again examined a few days
later, when the solutions had become darker.
Amino-compound used. Colour produced.
Glycine value = 8 red+13 yellow.
Alanine Moor Gy tak et tlie Wape rs!
Leucine iia eae loci Oe 2h
nenivglalanine eagle none) tele
Tyrosine SN ro vewa tes Ayam
The three aliphatic acids give similar colours, the two aromatic acids
yield a somewhat different shade. Hence these pigments differ essentially
from those obtained by Chodat from p-cresol, inasmuch as the colours of the
latter depend on the nature of the amino-acid. Whatever be the explanation,
the formation of pigment from arbutin and protein degradation-products is
one which may well be of natural occurrence. In passing, it may be observed
that quinone, like alloxan and triketohydrindene, may prove to be of use in
the diagnosis of amino-compounds.
Substituted quinones such as 1:4-xyloquinone or 1: 4-thymoquinone
resemble quinone in giving a colour reaction with glycine on warming in
aqueous-alcoholic solution; but in the case of these substances the reaction
takes place much more slowly. Xyloquinone give rises to a claret red,
thymoquinone to a tawny or brown red. ‘There is apparently a difficulty in
reducing the quinones, as neither of them gives a colour reaction with
formaldehyde and ammonia.
Weare investigating the behaviour of other glucosides and find that salicin,
the glucoside of the willow and many other plants, gives an orange, passing
to an orange-red, coloration when hydrolysed by emulsin and oxidised by
an oxydase in presence of an amino-acid. Similar colours are obtained
with glycine and with phenylalanine, the tintometer reading in a half-inch
cell being in each instance 4:5 red + 1°8 yellow. Salicin incubated with
ground sweet almond and a few drops of hydrogen peroxide gives a similar
colour reaction.
Phloridzin, the glucoside present in the roots of many rosaceous trees, is
composed of glucose and phloretin, a condensation product of p-hydroxy-
hydratropic acid and phloroglucinol. When hydrolysed by emulsin in
K 2
124 Prof. Keeble, Dr. Armstrong, and Mr. Jones.
presence of glycine it is converted into a yellow substance which becomes
orange and finally orange red. A red insoluble deposit, which separates out,
forms an orange-red solution in alcohol. In the tintometer we find for
a t-inch cell: alcoholic extract, 3°5 red+1:5 yellow; aqueous solution,
2°5 red+5 yellow.
Finally, zesculin (from the horse-chestnut) gives a yellow precipitate, and
aucubin (from the red berries of Aucuba japonica), a black precipitate under
the conditions described.
The property of colour formation from a glucoside and an amino-acid seems
to be a very general one, though we are unable to say whether the mechanism
is in each case the same as we have postulated for arbutin, namely, oxidation
of the phenol to a quinone, formation of a quinohydrone and SMS DE of
this with ammonia to form a coloured salt.
In any case, Chodat’s discovery of the resolution of amino-acid into
formaldehyde and ammonia is obviously of fundamental importance. The
ammonia may serve to provide the alkaline conditions so favourable for
oxidation and it may react directly to form amino-compounds. The
formaldehyde may take part in all manner of condensations leading to the
production of complex substances.
Section 3.—The Biochemistry of Mendelian Colour Characters.
Of the various artificial chromogens which serve to determine the presence
of oxydase in flowers, benzidine behaves most like the natural chromogens.
For example, most artificial chromogens, «-naphthol, guaiacol, ete., serve well
enough to indicate the presence of oxydase in the vascular tissues (veins),
but they do not react as a rule with the oxydase contained in the epidermal —
cells, whereas benzidine gives uniformly good reactions with both epidermal
and bundle oxydase (see Part I). Again, just as the reducing agents present
in petals may reduce the anthocyan pigments to a colourless state, so these
same agents reduce and decolorise the blue oxidation product of the
interaction of plant oxydase and benzidine.
Inasmuch as benzidine has proved to be of considerable value for the
investigation of plant oxydases, it may be useful to preface this section with
a brief account of what is known of the oxidation products of benzidine,
which are of an unusually complex character.
Willstatter and Kalb* have shown that the first oxidation product of
benzidine (NH2CgHsCsHsNHe) is probably the reddish-brown dipheno-
quinone di-imine NH:CsHu:CsHs:NH. On further more drastic oxidation,
two molecules of this substance unite to form the yellowish-red diaminoazo-
* “ Ber.,’ 1905, vol. 38, p. 1232.
The Formation of the Anthocyan Pigments of Plants. 125
diphenyl NH2.CgH4.CeHy.N:N.CgH4.CeHs.NH2. The blue- and violet-browns,
so characteristic of the action of oxydases on benzidine, are due to complex,
partially or meri-quinonoid salts of diphenoquinone di-imine with benzidine
itself :-—
NH:C,Ha:CsHa:NH = Quinone diimine.
NH>.CgHy.CgHy.NHe = Benzidine.
The molecules are united through the partial valencies of the nitrogen
atom. These compounds are meri-quinonoid, in that the quinone di-imine
may be combined with several molecules of benzidine. For example,
Willstatter and Piccard* describe a blue compound of the di-imine with
four molecules of benzidine, and a brownish-violet compound with three
molecules of the amine. Upon reduction, such meri-quinonoid compounds
are converted into benzidine, whilst oxidation transforms them gradually
into the quinone di-imine, as more and more of the benzidine is oxidised.
For the investigation of plant oxydase, and of inhibitors of oxydase, we
find that it is convenient to use benzidine in two forms, viz., a 4-per-cent.
solution in 50-per-cent. alcohol, and a saturated solution in 1-2 per cent.
of sodium chloride.t When rapidity of action is required, the latter solution
is employed, but, when inhibition is under investigation, the alcoholic
solution should be used side by side with the sodium chloride solution. In
illustration of the rapidity of action of the sodium chloride benzidine
solution, it may be mentioned that, if young seedlings of maize, etc., or
mature roots of water plants such as Hydrocharis morsus-rane (frog bit) be
immersed for a few minutes in this solution, the subsequent addition of a
few drops of hydrogen peroxide causes almost instantaneously a bright blue
coloration of their root-hair regions.
Again, if flowers known to contain an inhibitor of oxydase be treated with
some agent, for example, absolute alcohol, which is known to remove the
inhibitor (see Part IV), they fail to react with the alcoholic benzidine
solution until the whole or greater part of the inhibitor has been removed,
whereas such flower sgive a definite reaction with sodium chloride benzidine,
even though the inhibitor has been only in part removed.
The subject with which we deal in this section is that of the cause of the
range of flower-colour which occurs within a species. In illustration of
the nature of this problem we may mention the facts known in the case of
the flowers of the chinese primrose (Primula sinensis). In addition to white-
* “Ber.,’ 1908, vol. 41, pp. 1458, 3245.
+ Cf. Madelung, ‘ Zeitsch. physiol. Chem., 1911, vol. 71, p. 204.
126 Prof. Keeble, Dr. Armstrong, and Mr. Jones.
flowered races (dominant and recessive whites), the horticultural varieties
of this species comprise races with blue, red, and magenta flowers, and our
purpose is to put forward a biochemical hypothesis to account for the
production of these distinct colours and for the genetical relations which
obtain between them.
It has been suggested by Miss Wheldale* that each of the chief colours of
such a series is determined by a special oxydase, but neither general
considerations nor such observations as we have been able to make lend
support to this view.
It is true that the flowers of different varieties of P. sinensis contain
different amounts of oxydase, but we find no constant relation between
amount of oxydase and type of coloration. Moreover, the recent researches
of Bacht point definitely away from the hypothesis that oxydases are specific.
If hypothesis of specific oxydases be rejected, we must ascribe specific
coloration either to the action of an oxydase on different chromogens or to
the interaction, with a chromogen or an oxydase, of specific substances which
modify decisively the colour produced in the course of the reaction.
Any discussion of these alternatives must take into account the observations
of A. G. Perkin, that the hydroxyflavone glucosides of plants occur, as a rule,
not singly but in groups. There is some ground for regarding these glucosides
as constituting the prochromogens from which the anthocyan chromogens are
derived, and it is therefore a matter of great significance to the student of
genetics that the plant is, as it were, offered a choice of several pigment-
forming materials on which its hydrolysing and oxidising enzymes may act.
Pending fuller investigation of the possibility that the colour of a variety
may be determined by a selective action on one of a group of allied glucosides,
we are inclined to adopt the latter of the two alternatives, and to suggest that
the serial colours of flowers are due each to the intervention of specific
substances in the reaction of oxydase on chromogen.
This hypothesis is rendered plausible by the following observations, first
on the colours produced when a mixture of phenols is treated with oxydase
and second, on the behaviour of our artificial chromogen benzidine when
acted on by oxydase in the presence of various phenols.
When a mixture of phenols is treated with a plant oxydase a competition
for oxygen ensues. For example, if oxydase be caused to act on guaiacol
until the red colour is produced, the addition of other phenols brings about
a more or less quick change of colour. Thus a-naphthol converts the red
into mauve, and the ultimate colour which is produced is of a far deeper tint
* “Prog. Rei Bot.,’ 1910, p. 469.
+ ‘Arch. Sci. Phys. Nat.,’ June, 1912, vol. 33.
The Formation of the Anthocyan Pigments of Plants. 127
than that which arises when z-naphthol and oxydase interact with one
another. If p-cresol be added to the red solution produced by the action of
oxydase on guaiacol a brown colour appears. Saligenin, on the other hand,
does not modify the normal deep red colour given by guaiacol and oxydase.
Search in chemical literature brings to light a few records of similar
observations. Thus Schoenbein, in 1856,* observes that guaiacum blue
oxidises other oxidisable substances, and in doing so becomes reduced and
decolorised. Kastle and Porcht+ find that the oxidation of p-phenylene
diamine, guaiacum, and phenolphthalein, by means of an oxydase, is accele-
tated greatly by phenol, the cresols and B-naphthol. They recognise that
these accelerators act probably as auxiliary oxygen carriers, and that
they are themselves more or less completely oxidised in the process.
Miss Wheldale? suggests that oxidised catechol acts as a peroxide.
p-Phenylene diamine (p-diamino-phenyl, NH2CsH:NH2) exhibits a some-
what different behaviour. Together with z-naphthol it constitutes the
indophenol reaction for oxydase, which reaction is used largely by animal
physiologists. In it the oxidised amine and phenol are coupled to an
indophenol.
We find that phenylene diamine gives much the same violet-blue
coloration with all phenols, including methyl quinol; but that benzidine
and oxydase give with each phenol a distinct colour, similar to that produced
when the phenol in question is oxidised by oxydase. It would appear,
therefore, that nothing analogous with the indophenol reaction takes place
when benzidine and phenols are acted on by oxydase.
It is to be noted that p-phenylene diamine is oxidised by atmospheric
oxygen to a garnet red (tetra-aminodiphenyl-p-azophenylene), whereas it
gives a dark brown product when oxidised by oxydase. The indophenol
reagent is ill-adapted for the localisation of plant oxydases because of the
readiness with which it oxidises spontaneously. Petals of recessive white
Primula sinensis give a general purple reaction with it, and a brown with
phenylene diamine, but solutions of these reagents become strongly coloured
even without the addition of hydrogen peroxide, whereas this is not the
case with benzidine and the other reagents which we use for the investigation
of plant oxydases.
We have not used as-dimethyl-p-phenylene diamine (NH,;CsHsNMe.),
which substance is oxidised readily to quinone imine, the salts of which
* ‘Journ. Prakt. Chem.,’ vol. 57, p. 496.
+ ‘Journ. Biol. Chem.,’ 1908, vol. 4, p. 301.
t ‘Roy. Soe. Proc.,’ 1911, B, vol. 84.
128 Prof. Keeble, Dr. Armstrong, and Mr. Jones.
form red meri-quinoid compounds with the unchanged diamine—the so-called
Wurster salts. :
We have noted already that when hydroquinone is added to a mixture of
benzidine and oxydase in which the blue colour has been allowed to develop
the colour is discharged. It is not until all the hydroquinone has been
oxidised that the blue colour begins to return, the limiting factor being the
amount of hydrogen peroxide present. Most other phenols behave similarly
to quinol, but since their oxidation products are generally coloured, the blue
benzidine mixture becomes colourless for an instant only and then the solution
assumes a lavender, green, red, or brown hue, according to the phenol chosen.
This colour slowly changes, and as the benzidine blue returns it becomes
masked, and finally overpowered by the blue.
The phenols experimented with include p-cresol, orcinol, guaiacol, «- and
8-naphthol, thymol, pyrogallol, resorcinol, phloroglucinol, saligenin, phenol,
methyl quinol, dimethy] quinol, etc.
The list comprises certain phenols which ally do not give a colour
reaction with oxydase, ¢.g., methyl quinol. Even with «-naphthol the normal
lavender oxidation coloration is much more intense when produced in the
presence of other phenols.
The behaviour of methyl quinol deserves special mention in that it affords
the basis for our hypothesis as to the production of serial colours in flowers.
With oxydase, methyl quinol gives no colour reactions; but if a little
benzidine be added to the colourless solution the latter takes on a deep
and persistent carmine colour. The blue benzidine pigment acts catalytically
as an intermediary for the transmission of oxygen to the methyl quinol ;
that is, it may in this respect, and in this case, play the part of an organic
peroxide, and thereby achieve the oxidation of a substance (methyl quinol)
which resists the action of oxydase and hydrogen peroxide.
The power of benzidine to transmit oxygen to methyl quinol and other
phenols may be illustrated by making use of the oxydase present in the
flowers, or other parts of plants. For instance, if the flower of a recessive
white P. sinensis be treated with benzidine and hydrogen peroxide, the petals
assume the blue-brown colour characteristic of the benzidine-oxydase reaction.
If, however, benzidine and methyl quinol be added together with hydrogen
peroxide a carmine coloration is produced.
A similar oxygen transmitting power on the part of benzidine is exhibited
in the behaviour of the white flowers of Lychnis coronaria. Treated with
benzidine alone the petals become brown; with «#-naphthol they take on—
albeit with extreme slowness—a lilac or lavender colour. If, however, the
petals be treated with benzidine and «-naphthol they assume immediately a
The Formation of the Anthocyan Pigments of Plants. 129
lilac colour which, taken in conjunction with the previous observations,
indicates that benzidine facilitates the transference of oxygen from oxydase
to a-naphthol.
In order to make clear the closeness of the analogy between the oxydase-
benzidine and oxydase-benzidine-methyl quinol reactions on the one hand and
those which lead to the production of the quinol colours—blue, red, and
magenta—of such a plant as P. sinensis, it 1s necessary to give a brief account
of the genetics of flower colour in this plant.
The flowers of P. sinensis stand in a definite and constant relation with
one another. They form a series: recessive white, blue, red, magenta and
dominant white. The biochemical nature of the whites has been described in
an earlier communication (Part III).
Of the coloured members of the series blue is recessive to both red and
magenta, and red, which is dominant to blue, is recessive to magenta.
The Mendelian interpretation which fits the genetical facts is as follows :—
The character for blue flower depends on the presence of a single
Mendelian factor. Red flowers also contain this factor and they contain in
addition a factor for red which can produce its effect only in the presence of
the “blue” factor. Similarly magenta flowered plants contain a magenta
factor which when present together with the red and blue factors gives rise
to the magenta character.
In the absence of the lower members of the series, colour is not produced
and the colour of any flower is an indication that the series of factors is
unbroken up to the factor for the colour character manifest in the flower.
We have thus a striking parallel between the colour series in P. sinensis
and that which occurs with benzidine and methyl quinol. The closeness of
the parallel is indicated thus :—
P. sinensis. Biochemical model.
Oxydase. Oxydase.
N
Blue factor. + Red factor. Benzidine. + Methyl quinol.
V
Red. | | Red.
Blue. Colourless. Blue. Colourless.
The peculiar behaviour of the red factor, first in failing to induce colour
formation in the absence of the blue factor, and, second, in masking com-
pletely by a red pigment the activity of the blue factor, is to be accounted for
thus:—The red factor determines the formation of a specific substance—
perhaps of the nature of a phenol. That substance is not oxidised directly by
the oxydase of the flower, but in the presence of the “blue” factor this
specific substance receives oxygen from the blue pigment produced by the
130 Prof. Keeble, Dr. Armstrong, and Mr. Jones.
agency of that factor, and, in consequence, the blue pigment is reduced to the
state of a colourless chromogen. The observations recorded on p. 126 lend
additional support to this hypothesis. It is there observed that various
phenols intensify, though they may not change, the colour produced by the
action of oxydase on artificial chromogens. On the practical side it is also
known that intensifiers of pigment exist, that they possess the power of
converting a pale into a deep shade, and that they behave each as a unit
character. On our model it seems reasonable to assume that an intensifier is
a phenolic or similar substance, and that the factor for an intensifier means
the power of the cell to produce that substance.
Lastly, on the basis of this hypothesis we have a plausible explanation of
the fact that many oxydase reagents, though they give good “bundle”
reactions, fail to reveal the presence of oxydase in the epidermis. The
vascular tissues contain considerable stores of oxydase and oxygen-carrier,
and hence, through the agency of the carrier, oxygen is transferred to.
e-naphthol or similar “artificial chromogen.” The epidermal tissue contains
only a small quantity of the carrier of oxygen, and hence, in spite of the
presence of oxydase, «-naphthol and similar artificial chromogens remain
unoxidised in this tissue.
Conclusions.
1. The pale yellow sap colour of the petals of the wallflower is a mixture
of hydroxyflavone glucosides. The glucoside mixture is hydrolysed readily
by heating with mineral acids and more slowly by emulsin of almonds. The
hydrolysed product if reduced and subsequently oxidised yields a red
pigment.
2. The fact that flowers containing similar soluble yellow pigments may be
caused, by suitable chemical treatment, to yield a red pigment, suggests that
red mutations should be of possible occurrence in such species.
3. The formation of pigments, as the result of oxidation by oxydase of the
hydrolysed products of glucosides, is determined by the presence of amino-
compounds and is of very general occurrence. The behaviour of the glucoside
arbutin (see p. 121) makes it probable that many of the pigments and odorous
substances formed during the ripening of fruits arise as results of reactions
of this type.
The pigments of plants may be classified provisionally as follows :—
I. Plast Pigments—
a. Chlorophyll prementsicomtaim pee-eeeeeenencer emcee ries C, H, O, N
ig), Ob wetoy merle) COMMIS! Br. gacnsuecaccbbagceccs 20 ;oceouannde2035 Jel
The Formation of the Anthocyan Pigments of Plants. 131
Il. Sap Pigments—
a. Yellow. Hydroxyflavone glucosides or derivatives
UINEIREON! COMRTIN, 3 op baresrisaonnopanbaorineace ancodaseeecoce CAO
b. Red, ¢.g., of wallflower (see p. 117). Products of the
action of oxydase on hydroxyflavone glucoside
GETIVALLVES | COMLANME pray oa celajot tere oe os cea tedancelne ones CeHy@
[ Whether all anthocyan pigments are of this type is
unknown. |
c. Red and brown, ¢g., of plum. Substances produced
by the oxidation of phenols in the presence of
ATMO) COMMMIIG —.saacenoonesodeastescasocaedeobads CAH OMN
d. As suggested in Section 3, the so-called anthocyan
pigments (red and magenta) of flowers may arise
as the result of the oxidation of phenol brought
about by an organic oxygen carrier ; contain ...... ©, H;,0
4. The benzidine-methylquinol-oxydase reaction (p. 128) provides an
analogy with the Ild type of pigment formation, and suggests the hypothesis
that the higher members of a flower colour series (see p. 129) owe their origin
to the presence with the lower members of specific substances which, acting
as receivers of oxygen, reduce the pigments characteristic of the lower
members of the colour series, accept oxygen, therefrom, and thereby become
oxidised to pigments of specific colour.
132
On the Question of Fractional Activity (“All or None”
Phenomenon) in Mammalian Reflex Phenomena.
By T. GRAHAM Brown (Carnegie Fellow).*
(Communicated by Prof. C. 8. Sherrington, F.R.S. Received June 17,—
Read June 26, 1913.)
(From the Physiological Laboratory, University of Liverpool.)
CONTENTS.
PAGE
Toe) dmtrodactlont jes es neuer scsecceamececc sas 132
II. The Experimental Evidence................+. 132
III. Objections to “all or nothingness” ...... 134
IV. Methods here employed .............c2sc000+ 136
Wa! WResullts 5.15.25 vacmelates beseegecuactuseaaccessserncs 137
Wale (Conclusionstietnr-wsssccceeseccestecassweaseccen ne 139
VALE eS UmmiaMys! sveadesstoreecrsncescer sur eereenene 141
I. Introduction.
At the present moment the question whether or not there is a state of “all
or nothing” activity in reflex arcs seems to be raised, and it is one of
importance to the future of investigation of the functions of the nervous
system.
Of the two views which may be held regarding the manner of the activity
of reflex arcs one is that in which it is supposed that the efferent neurone
may react with different degrees of intensity in different reflex activities, and
that the afferent neurones may play with different degrees of intensity upon
efferent neurones or upon interposed neurones.
The other view, which seems now to be dawning, is one in which it is
supposed that the efferent neurone has no grading in the intensity of its
activity—it either reacts maximally or not at all; and if this be demon-
strated it may perhaps be inferred that the afferent neurones act in a similar
manner—that is, that their activity is either “all or none.”
It is obvious that, if either of these views is shown to be the correct one,
the course of research will in the future be modified.
Il. The Experimental Evidence.
There is, at present, little direct experimental evidence bearing upon the
question of “all or nothing” activity in reflex arcs.
* The expenses of this research have been defrayed by a grant from the Carnegie
Trust.
Fractional Activity in Mammalian Reflex Phenomena. 133
But in 1902 Gotch* showed that the electric organ of Malapterurus, which
is served by a single efferent fibre, as compared with that of Torpedo, which is
served by many fibres, has a much smaller range of reactions to reflex
excitation. He yet states that within very restricted limits the organ shock
is slightly augmented when an effective stimulus applied to its nerve is
increased in intensity. He also notes that the initial shock reflexly evoked
is variable in intensity, but he states that a considerable factor in this
variation of intensity is probably fatigue of the nerve endings in the organ.
There is, however, some evidence which seems to show that the efferent
nerve fibre and the effector organ (skeletal muscle) when stimulated
artificially by faradic shocks may respond in an “all or nothing” manner to
the peripheral stimulus.
In 1905 Keith Lucast showed that when the exciting current which is
applied to the skeletal muscle of the frog is gradually increased in strength
the contraction of that muscle increases, not pari passu, but in abrupt steps.
Four years later the same investigator showed{ that in the frog’s cutaneus
dorsi muscle, there is an “all or nothing” contraction of the muscle fibres in
response to stimulation of the efferent nerve fibres—submaximal contraction
of the whole muscle being due to a maximal contraction of less than the
whole number of constituent muscle fibres.
Vészi, in 1911,§ made a curious observation which seems to throw doubt
upon the “all or nothing” phenomenon in reflex conduction. He found, in
the de-afferented frog, that in the state of strychnine poisoning there is an
“all or nothing” contraction of gastrocnemius in response to stimulation of
the cut afferent roots. A threshold stimulus evokes a maximum muscular
Tesponse. But when this has fatigued by repetition of stimulation, a stronger
stimulus again gives a maximum response.
In the following year the same observer|| came to the conclusion that the
fresh amphibian efferent nerve-fibres follow the “all or nothing” rule; but
that when fatigued the value of the excitatory process varies with the value
of the exciting stimulus.
Slightly more recently Adrian{i has investigated this question. He finds
that a propagated disturbance in the efferent nerve fibres of amphibian nerve
which has been reduced in magnitude by passing through a region of
* ¢ Journ. Physiol.,’ 1902, vol. 28, p. 395.
+ ‘Journ. Physiol.,’ 1905, vol. 33, p. 125.
t ‘Journ. Physiol., 1909, vol. 38, p. 113.
§ ‘Zeitschr. fiir allgem. Physiol.,’ 1911, vol. 12, p. 358.
|| ‘Zeitschr. fiir allgem. Physiol.,’ 1912, vol. 13, p. 321.
“7 ‘Journ. Physiol.,’ 1912, vol. 45, p. 389.
134 Mr. Graham Brown.
decrement regains its original size when it emerges into normal tissue. He
points out that this favours the supposition that the relation between the
disturbance and the strength of the evoking stimulus is of an “all or
nothing” character.
Quite recently Mines* has given a description of experiments which suggest
either that there is no gradation in the response of efferent amphibian nerve
fibres to graded stimuli consisting of single induction shocks, or that the
smallest excitation is capable of exciting any neuro-muscular synapse which
can be excited by single impulses.
These various experiments certainly seem to point to the conclusion that
the response both of the peripheral efferent nerve fibres and of the muscle
fibres of the skeletal muscles is of an “all or nothing” character when the
exciting stimulus is an artificial electrical one. And there is a temptation
to argue from this that the activity of the same efferent nerve fibres and
skeletal muscle fibres in the less artificial reflex excitation is also of an
“all or nothing ” character.
If this view be taken we must suppose the efferent neurone to discharge
maximally or not at all. We must look at the reflex mechanism as one
split longitudinally into units (as indeed we do look at it), which are each
elther maximally active or inactive, but never of intermediate activity. We
must suppose that the grading of the muscular response is due to the differing
proportions of its component units which at any one time are in action.
But we must even then admit that a certain sort of grading of activity may
occur even in one efferent neurone—for it might be supposed that the
discharges proceeding from it might vary in frequency.
A subsidiary question is that of the possibility of a similar “all or nothing”
character in the activity of the afferent neurones.
ILL. Objections to “ All or Nothingness.”
At first sight it might seem that a strong objection to the “all or none”
character of afferent activities is before our eyes on any clear and moonless
night. The stars appear to be of very different brightness, although the
size of their images upon the retina is almost infinitely small and, theoreti-
cally at any rate, must be looked upon as stimulating only one retinal
element each. It might seem that it is hardly possible to explain the
number of distinguishable brightnesses as due to different numbers of retinal
elements stimulated—for instance, are as many as ten stimulated in the
case of a bright star, and as few as one in a star on the limit of visibility ?
* “Journ. Physiol.,’ 1913, vol. 46, p. 1.
Fractional Activity in Mammalian Reflex Phenomena. 135
Plausible as this objection to the “all or none” character of a certain
specific afferent activity seems, it must be remembered that the imperfections
of the eye may condition irradiation and halation sufficient to allow of the
explanation of the phenomenon on the “all or nothing” principle.
But again, a wide range of touch sensations of different intensities may
be obtained on stimulating the one point on the skin with stimuli of different
values—and this even when the area of skin stimulated is supplied by a
single afferent nerve. Here there seems to be no “all or none” response to
eraded stimulation.
Yet again, Sherrington* found for the scratch-reflex of the spinal dog that
a dozen or more grades in the reflex response might be obtained on graded
punctiform stimulation of the same skin-point—that is, of the same afferent
nerve fibre.
These two latter observations seem to point very strongly to the conclusion
that there must be a grading of the response, at any rate, in certain varieties
of afferent nerve fibres of the mammal in reply to graded intensity of
stimulation. And when we examine the possibility that the reflex discharge
of the efferent neurone is of an “all or none” character certain difficulties
are presented.
In the first place we have Sherrington’st statement that in a reflex
response of the muscles of the hind limb of the mammal all the contractors
are active in the minimal reaction, and that grading of the intensity of the
reaction In response to grading of the intensity of the stimulus is accom-
plished by an increase in the activity of each contractor. If, however, the
activity of each individual contractor is conditioned by a fractional activity
of its fibres, it is, at any rate, strange that the thresholds of the most excitable
fractions should be the same in all the contractors.
Secondly—perhaps not a very grave difficulty—there is the question of
“tonus.” This slight contraction on the “all or none” theory must be
looked upon as due to the activity of a few of the muscle fibres only. But
no sagging or wrinkling is seen in parts of a tonically contracted muscle,
and the pull of the muscle where the tendon is a broad one is not seen more
at one side than another.
Thirdly—and a more formidable difficulty—the “beats” of the scratch-
reflex are usually “incomplete.” That is to say, the flexor (for instance)
exhibits a series of partial relaxations and reconstitutions of contraction.
These may be looked upon as conditioned by a series of incomplete refractory
phases. Now under an “all or nothing” theory each partial relaxation must
* * Journ. Physiol.,’ 1906, vol. 34, p. 1.
+ ‘Journ. Physiol.,’ 1910, vol. 40, p. 28.
136 Mr. Graham Brown.
be looked upon as produced by the “complete” relaxation of a proportion
of the muscle fibres—or by the “ complete ” inhibition of a proportion of the
efferent neurones. But Sherrington* has shown that the refractory phase
extends over the whole centre—just as Zwaardemakert showed the same
phenomenon for deglutition. As regards this, the centre is therefore a unit,
and in some manner innervated as a whole from each afferent neurone. It
is, however, impossible to suppose that each afferent comes into direct
contact with each efferent in the mechanism; and under any other supposi-
tion it is extremely difficult to realise the meaning of this unity if the
efferent discharges have an “all or none” character,
IV. Methods here Employed.
If a skeletal muscle exhibits a larger number of degrees of contraction
than there are efferent nerve fibres running to it, there must be a very
strong supposition that the reflex response has not an “all or none”
character. In such a case the deduction from the result would not need to
be influenced by the number of afferent fibres stimulated.
Unfortunately, the large number of efferent fibres which supply most of
the skeletal muscles makes this experiment in their cases impossible.
In the cat, however, a most beautiful muscle in the hind limb—tenuissimus
—seems almost to have been made for this experiment.
This muscle is a thin band of only 2 or 3 mm. in breadth, but many
centimetres long. It arises from the caudal vertebre at the root of the tail,
and passes down deep in the thigh, until it ends in the leg by blending with
the insertion of biceps. For the upper part of its length it lies near the
great sciatic nerve, from which, near its middle, it receives its nerve supply.
Occasionally it receives more than one nerve twig, and, in any case, its chief
nerve divides into two branches (occasionally into more than two) before
it reaches the muscle. The number of nerve fibres in each of these branches
is small.
The cats used were decerebrate and low spinal. All the muscles of the
left hind limb were destroyed by motor paralysis. In the right hind limb
all were destroyed save tenuissimus. The great sciatic nerve was ligatured
after it had divided into external and internal popliteals. The biceps muscle
was divided transversely to its length about the middle, and tenuissimus
was thus exposed.
All but the uppermost of the branches of its motor nerve were severed,
* ‘Journ. Physiol., vol. 31, ‘Physiol. Soc. Proc.,’ March 19, 1904; ‘Journ. Physiol.,’
1906, vol. 34, p. 1.
+ ‘Archives Internat. de Physiol.,’ 1904, vol. 1, p. 1.
Fractional Activity in Mammalian Reflec Phenomena. 137
and the muscle was cut across a little below the point where its nerve
reached it. A fine silk thread connected it to the recording lever. This
was a fine heart lever pivoted on agate cups.
A fine silk thread was placed loosely round the intact branch of the
motor nerve. Electrodes were placed on the great sciatic nerve, as far as
possible peripheral to the point where the motor nerve left it for tenuissimus.
All the exposed surface was then covered, but tenuissimus itself was left free
and uncovered.
The reflex stimulus—faradic shocks, 30 per second—was applied for one
second every minute. The mechanical responses were recorded upon the
slow drum, but sometimes every 10th response was recorded on a faster
drum. The intensity of stimulation was varied by changing the angle
between primary and secondary coils degree by degree; sometimes also by
sliding up the secondary millimetre by millimetre. In some cases the
strength of stimulation was increased from a minimum, sometimes it was
decreased from a maximuin. Between stimuli the muscle was carefully
covered up.
V. Results.
The observations here described rarely lasted for less than an hour—during
which time a reflex contraction was recorded every minute. In these
circumstances it was found that a-certain deterioration of the preparation
occurred—so that a direct muscular contraction was smaller at the end of
the series than was one taken with the same strength of stimulation at the
commencement of the series.
If a series of reflex contractions commenced with strong stimuli, and was
continued with ever weaker stimuli, it is probable that a grading due to
muscular “fatigue” might add itself to the true reflex grading. There
might appear a larger number of “ steps” than were actually conditioned by
a grading in the efferent nerve.
In these experiments, therefore, the reflex stimuli were varied in the
reverse order—that is, starting with subminimal stimul, and gradually
increasing the strength. Before and after each series a direct muscle
contraction was registered, and, as in every case the contraction was
smaller after the series, the number of different degrees of reflex muscular
contraction registered was possibly less than the actual number. That this
was so was also shown by the fact that, at the commencement of each
experiment, there was a greater range of reflex contractions in a “ quick”
series (that is, one in which the graded stimuli were of widely differing
intensity) than in the subsequent “slow” ascending series of reflex
VOL. LXXXVII.
B L
138 Mr. Graham Brown.
contractions, from which the estimation of the number of grades of
contraction was made.
At the end of each experiment a maximal reflex contraction was
registered. The remaining motor twig to tenuissimus was then divided, and
the same reflex stimulus was again applied. If there was no muscular
response it was assumed that that twig contained all the remaining motor
fibres, and these were counted after staining with osmic acid. The number
of different heights of reflex contraction was then counted and compared
with the number of nerve fibres. Differences in height of under 0°5 mm.
were neglected, and the heights were measured from the level of contraction
which obtained at the commencement of each reflex contraction.
The following table gives details of six experiments :—
No. Fibres. | Grades. | Kind of series.
i | 31-83 27 Ascending series.
2 24-27 47 Descending series.
3 42 40 Ascending series.
54 Descending series.
4 30 27 Ascending series.
5 48 28 (+21?) | Ascending series (broken).
6 28 31 Ascending series.
In Experiment 5 the series was taken in groups. Thus first a group of 10
closely graded stimuli was registered. The stimulus was then increased
10 times more than the increase between each pair of elements of this
group, and a second closely graded group was registered. Again the stimulus
was more greatly increased, and a third closely graded group was registered—
and so on. As in each group there were about seven different grades amongst
the 10 contractions, and as there were three “gaps” it is reasonable to
suppose that about 21 grades should be added. The difference in height
between the end contraction of a group and the commencing contraction of
the next group was nearly the same (being sometimes greater) than that
between the initial and terminal contractions of a group.
It will be observed that there are more grades in a descending series than
in an ascending one. If the least favourable kind of series—that is, the
ascending—be taken, it is found that the number of grades of reflex con-
traction corresponds fairly closely with the number of nerve fibres in the
efferent nerve. It is sometimes a few more and sometimes a few less.
If this be the case, then there are more grades of reflex contraction than
there are efferent nerve fibres.
For, in the first place, the presence of deterioration of the motor response
Fractional Actunity in Mammalian Reflec Phenomena. 139
probably means that there should have been more grades than were actually
recorded.
And secondly, not all the fibres in the efferent nerve are efferent nerve
fibres. A certain proportion of them are afferent nerve fibres from the
sensory end-organs in the muscle. If tenuissimus conforms to the ordinary
rule, about two-thirds to one-half only of the fibres in the motor nerve are
efferent—for that is the proportion which Sherrington* found for the
monkey and cat.
VI. Conclusions.
The experiments here described seem to show that the number of different
mechanical responses with which a specific individual muscle (a flexor)
answers certain reflex stimuli (ipsilateral flexion-producing) of different
intensities may be greater than the number of efferent fibres in the motor
nerve. ‘The differences in mechanical response are here measured by
estimating the extent of the greatest shortening of the muscle during a reflex
tetanus which lasts 1 sec.
On the assumption that each difference in shortening of a greater extent
than 0°5 mm. in the tracing as magnified by the lever is a measure of a
different degree of reflex activity, it would seem that the activity evoked
reflexly in the efferent nerve fibres here investigated has not an “all or
none” character ; and that the discharge of any efferent neurone may be
graded in resonance with graded afferent stimuli.
But it must be admitted that the mechanical response is a coarse indicator
—even under the conditions here used. And it must further be admitted
that even if there is a larger number of mechanical responses than is the
number of efferent nerve fibres this does not of necesssity exclude the
possibility of an “all or none” character in reflex efferent discharges.
It is possible, for instance, to look upon the efferent part of a system as
composed of three longitudinal parts—A, B, and C—each of which consists
of efferent neurone and subservient muscle fibres. Three graded afferent
stimuli might discharge: the first, A; the second, A+B; the third,
A+B+C. In such an arrangement it would be expected that there would
be three distinct and separable grades of mechanical response. But it is
also possible that a series of graded afferent stimuli might discharge: the
first, A; the second, A+B; the third, A+C (but not B); the fourth, B+C
(but not A); the fifth, A+B-+C. In such a case there would, therefore,
be five possible mechanical responses if the muscular elements were of
different efficiencies. This is certainly a possibility, but it must appeal to us
at present as being too artificial a possibility.
* ‘Journ. Physiol., 1894, vol. 17, p. 211.
140 Mr. Graham Brown.
It seems best at present, in view of the difficulties met with in assuming
an “all or none” activity, at any rate, in certain species of reflex ares, to
hold that the efferent neurones may discharge each with graded intensities.
If that be the case then it must appear that there is an essential difference
between the activity cf efferent nerves aroused by artificial peripheral stimuli
and those evoked reflexly through the centres.
The question must arise whether this grading is one in which the
amplitude of the discharge of each neurone may be varied, or whether the
grading is produced by different speeds of repetition of discharges, the
amplitudes of which are not varied.
In the latter case an explanation is offered only if the mechanical response
varies with variation in the speed of repetition of nerve impulses.
That this is indeed the case Mines* has recently given some evidence to
show. He points out that the ordinary explanations of the greater tension
produced during tetanus than in single muscular twitches do not meet the
case. He notes, for instance, that the fact that the tension set up in
amphibian muscle in response to more rapid stimuli is greater than that set
up in response to less rapid stimuli (which yet are sufficiently rapid just to
give complete fusion) is not explained on the von Frey hypothesis.
One more point. The aspect of the problem which here particularly
interests us is the question of an “all or none” response of the efferent
neurone to graded reflex stimuli. Even in the case of peripheral stimulation
there is little or no evidence of an “all or none” character of the response
to graded stimuli of the efferent nerve fibre considered as a unit. As
Adriant himself points out, his experiments seem to show that certain
longitudinal units of conduction are characterised in their activity by an
“all or none” response to graded stimuli, but there is nothing to show that
these units are the nerve fibres.
If they are units of a smaller size than the nerve fibres the efferent
neurone may still respond in a graded manner to graded stimuli, although
the activity of the elements of the discharge may be distinguished by this
“all or none” character. If this be the case the discharge of the efferent
neurone might be graded in “steps” from zero to its maximum.
That either the reflex discharge of the efferent neurone has not the
character of an “all or none” response to graded stimuli, or that the longi-
tudinal units, the activities of which possess the character of an “all or none”
response to graded stimuli, are smaller than the nerve fibre seems to be
shown by the experiments here described for one specific reflex type.
* ‘Journ. Physiol.,’ 1913, vol. 46, p. 1.
+ ‘Journ. Physiol.,’ 1912, vol. 45, p. 389.
Fractional Activity in Mammalian Reflex Phenomena. 141
VIL. Summary.
The mechanical response of tenuissimus—a flexor in the hind limb of the
cat—to graded reflex stimuli (tetani, lasting one second) seems under certain
conditions to exhibit grades of difference greater in number than the number
of efferent fibres in the motor nerve which supplies it.
On the assumption that the differences here observed denote differences
in the activity of reflex discharges, this seems to show that the discharge of
the efferent neurone in a specific type of reflex activity has not the character
of an “all or none” response to graded stimuli.
This does not, of course, exclude the possibility that within the neurone
there are units, the activities of which have this character.
Experiment 24.10.12 (No. 5 in table). Decerebrate cat; a record of the
mechanical responses of right tenuissimus obtained in response to graded
reflex stimulation of the right great sciatic nerve. Cat decerebrated
10.45 AM.
The series was started at 11.27 4.m. The reactions are obtained in response
to tetani lasting 1 sec. (rate of stimuli 30 per second), and they are taken
every minute. The electrical stimuli are graded at first—with the primary
and secondary induction coils 150 mm. apart—by rotating the secondary coil
and thus diminishing the angle between its axis and that of the primary by
1° for each reaction. Later in the series the electrical stimuli are graded by
pushing the secondary coil 1 mm. nearer the primary for every reaction (the
axes of the coils then are parallel). The series is broken into groups of 10,
and between the groups the electrical stimulus was graded tenfold the grading
between the elements of the group by 10° or 10 mm. Between the five final
reactions the grading is also of this order. Beneath each tenth reaction the
value of the evoking stimulus (either in degrees divergence of the secondary
axis at 150 mm. distance between coils, or in millimetres distance of coils
with axes in line) is recorded.
In the first group there are at least eight different mechanical grades.
In the second group there are at least seven different mechanical grades.
In the third group there are at least seven different mechanical grades.
In the fourth group there are at least five different mechanical grades.
For the fifth group at least two more grades may be added. This gives
a total of at least 29 mechanical grades. It can hardly be doubted that about
the same proportion of grades would have been present in the first three
intervals. On the assumption that in each of these there were seven distinct
grades the total number of grades for the series would be about 50. “ Quick”
series registered before and after this record showed deterioration. This
Graham Brown.
Mr.
142
deterioration probably hinders the number of evident grades of contraction in
such series.
‘L ‘eld
WW WW
ObL OGL
Fractional Activity in Mammalian Reflex Phenomena. 143
At the end of this experiment the remaining nerve twig of tenuissimus was
divided. Thereafter all reflex contraction was abolished and there were
48 fibres in the twig. Of these probably not more than 32 were motor.
MM
700 90 SO
<
S
~
Fia, 2.
In this experiment, therefore, a number of motor fibres which probably did
not exceed 32 conditioned reflexly a number of different mechanical grades of
contraction which probably did not fall short of 50.
144 Fractional Actuwity in Mammalian Reflex Phenomena.
Experiment, 28.10.12 (No. 6 in table). Low spinal cat, cord divided at
11 am. A record similar to that reproduced in fig. 1, save that the series is
complete. Series commenced at 12 o’clock, midday.
For the purposes of the experiment the following reactions may be con-
sidered to be of different grades: (marked by degrees) 1°, 0°; (marked by
millimetres) 149, 148, 145, 147, 141, 144, 143, 142, 140, 139, 138, 137, 135,
136, 134, 133, 132, 129, 130, 128, 127, 126, 123, 121, 117, 120, 118, 116, 100,
90 = circa 32 grades.
Before this series was taken a “quick” series showed greater grading—
between a minimum of 30° at 150 mm. and 90 mm. (coils in line). But in
the figure the grading is over a range of about 150 to 90 mm. only.
After the series here reprodueed was taken the remaining intact branch of
the motor nerve was ligatured. Thereafter no reflex contraction was evoked
on stimulation with the strongest stimuli here used. It was later found that
there were about 28 fibres in that branch of the nerve. Of these probably
about nine were afferent fibres.
Here, therefore, a number of grades which is probably about 30 was
conditioned by the reflex activity of about 20 efferent nerve fibres.
On Postural and Non-Postural Activities of the Mid-Brain.
By T. GraHAm Brown (Carnegie Fellow).
(Communicated by Prof. C. 8. Sherrington, F.R.S. Received July 21, 1913.)
(From the Physiological Laboratory of the University of Liverpool.)
CONTENTS.
PAGE
emlntroductiontrassmceeresssdsssterhccnc smectic: socheaten meee eccnseencsstistes sti 145
ieee Wet hodssPun ployed senecestea-eaessccdesteserasssaceeseecssanecsc aaepoaeapee 146
III. The State of the Monkey after Decerebration .................0.00eee0es 147
IV. Electrical Stimulation of Regions in the Cross-section of the Mid-
brain Dorsal to the Area of the Cortico-spinal Tract ............... 148
Ij the lpsilateralveaction s..ccc--sscscsee ne soeneecccecasce cer emee coace 148
J eeelivesContnalabenalsheachionersrctsenecrsecteten eerste cscs seecens 148
3. The Synchronous Compounding of Ipsilateral and Contra-
lateralineachlons™ eiesnccciscocesccenseceesmererrcene teeceteeteseess 149
4, The Compounding of Ipsilateral and Contralateral Reac-
HONG) tin ANE STNpEONAl! SWKEEER SION, peeconcconceqacsocéuerencooooocuenRe 154
5. The Geographical Position of the “ Focal Point” ............ 154
WereUhevbittectiotaVarious WWesiOns) vero.ssneccascscectccesecssceeceeneceses eastern. 155
1. Mesial Section between the Right and Left Halves of the
IGE A= Diratiiyeysststcnencusees sees citens ae nssebabstcceee asec as ene eee te oe 155
2. Right Semi-section of the Mid-brain between Anterior
an OP EOS tenior Colliculiteenmssadscecessedastetcccesemessteacceesies 155
3. Division of the Right Superior Cerebellar Peduncle......... 155
4. Complete Removal of the Cerebellum ..................0....060 155
& Tktermn@yyel Gre biG Els) dav soeougcpsconcsqccdeobeadaooquneoosccqoaados0000 156
VI. Electrical Stimulation of the Crus Cerebri.................scesesseseeeees 158
VII. Compound Stimulation of Crus and the more Dorsal Excitable
Area in the Cross-section of the Mid-brain ...............sseseceeeuee 158
1. Immediate Compounding of Crus against Contralateral
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2. Immediate Compounding of Crus against Ipsilateral
WeActiony (MIEXTOM)necneecesccmeaessececshnce scare ensenc seater ees 159
3. Compounding in Temporal Succession ..............:seeeeseeeees 159
VIII. Stimulation of other Points in the Mid-brain and Hind-brain ...... 159
XE CONCIUSIONS ee ssseeesstecdeccucsesccssoetscssacs eetpecesuidcasersensssucaestocsceises 161
I. Introduction.
In the course of experiments in which the cerebral cortex of the monkey
is stimulated, it is peculiarly noticeable that the activity of the cortex varies
from time to time. That such variation should occur is by no means
strange, in view of the difficulty of maintaining a constant depth of narcosis.
But there are other variations which seemingly are not conditioned by
146 Mr. Graham Brown.
variation of depth of narcosis. Thus it not rarely happens that, when the
depth of narcosis is certainly a constant one, the motor cortex becomes
suddenly inexcitable. This occurs, for instance, after a cortical discharge,
which is followed by “ epileptic” after-discharge. But it also occurs without
any apparent preceding cause. Thus suddenly the cortical excitability
becomes abolished—at any rate, to practicable strengths of stimulation.
This sudden loss of cortical excitability is a phenomenon of interest. It
is accompanied by two marked states. Of these, the first is an aneemia of
the cortex ; the second is a maintained postural contraction of certain of the
muscles of the limbs. The anemia seems to occur over the whole of the
small area of cortex—pre-central and post-central— usually exposed in these
experiments. It causes a sudden change in appearance from the “raw ham”
look of the cortex when it is in the most favourable condition for electrical
stimulation to a pale “dead” look. The cortex blanches; it may be surmised
that it faints.
The postural contraction of the muscles of the limb have most carefully
been examined in the case of the contralateral arm. In that member the
posture is one of flexion. The contraction of the flexors is a great one, and
it may be an exaggeration of the slight postural contractions (both flexion
and extension) which the arm always exhibits in changing degrees throughout
these experiments. But, from these muscular activities, this state of greater
contraction must be carefully distinguished. For, in the former, cortical
stimulation is effective, and can abolish or augment the postural contraction ;
but, in the latter case, the stimulation of the cortex is ineffective.
When this curious phenomenon is examined, two points stand clearly out.
There occurs a postural activity of the flexor muscles of the contralateral
arm, and this is accompanied by anemia and by inexcitability of the cortex.
The similar state of inexcitability which is seen after post-stimulatory
cortical epilepsy is also accompanied by blanching of the cortex, and often
by maintained posture of the arm.
From these facts the conclusion may be drawn that the postural activity of
the limbs in this state is conditioned by the activity of certain of the lower
centres. The question arises—which are these ?
Il. Methods Employed.
The animals used in the present experiments were small monkeys: Macacus rhesus,
Macacus sinicus, Callothrix, Cercocebus ethiops. They were kept unconscious throughout
the whole experiment, and until they were destroyed at its conclusion. The procedures
of decerebration, removal of the cerebellum, etc., were performed in the usual manners.
Stimulation of the various parts of the neuraxis was performed in the unipolar method.
Two unipolar electrodes were used in order that two points might be simultaneously
Activities of the Mid-Brain. 147
stimulated. These were on different circuits, and the two “ indifferent electrodes” made
necessary were applied one to either foot.
Stimulation of the peripheral nerves (ipsilateral and contralateral ulnars in the arm)
was performed in the usual bipolar method.
For the proper examination of the movements of the arm in these experiments the
movements of an extensor (humeral part of triceps brachii) and of a flexor (supinator
longus) of the elbow were registered simultaneously. All the other muscles of the left
arm and shoulder were destroyed by motor paralysis.
In the following descriptions the terms “ ipsilateral” and “contralateral” are used in
reference to the left arm—the former therefore meaning (here) “a point on the left side
of the body,” and the latter “a point on the right side.”
III. The State of the Monkey after Decerebration.
After comparatively high decerebration (that is, when the neuraxis is
divided across slightly anterior to the anterior colliculi), or even when the
division is through the anterior colliculi, the animal is not perfectly
immobile. When the depth of narcosis has shallowed the eyelids are open,
and sometimes wide open. Winking frequently occurs, and the eyes
sometimes are moved. From time to time the animal slowly changes its
posture, the movements being like those of normal sleep. Owing to the fact
that the animal in these experiments has been carefully covered and propped
in a definite posture for the recording of the arm movements, it is not easy
to describe those postures accurately. But, if the attention is confined to
the movements of the left arm, it is seen that slow postural flexion and
extension occur from time to time. The flexor thus may slowly contract,
and, having reached its maximum of contraction, may there remain for
many minutes if undisturbed. And, similarly, the extensor may at other
times contract and remain contracted. The hind limbs may shew postural
extension (Sherrington’s “ decerebrate rigidity”) and, although the state of
the hind limbs has not systematically been examined, it has seemed that
they tend more frequently to demonstrate the extensor rigidity than do the
fore limbs. From time to time the head is uneasily moved, and the animal
seems to react (by closing the eyes) to loud and shar 2 sounds—although my
evidence on this point is not very clear.
In short, the decerebrate monkey appears to be in a state which closely
resembles that of light sleep, and the fact that, in this condition, these slow
and maintained postural activities of flexion and extension in the arm may
occur after decerebration shews conclusively that they are conditioned by
centres below those in the cerebrum.
148 Mr. Graham Brown.
IV. Electrical Stimulation of Regions in the Cross-section of the Mid-brain
Dorsal to the Area of the Cortico-spinal Tract.
Unipolar stimulation of the cross-section of the mid-brain at the level of
the anterior colliculi—when applied at a point in an area which lies dorsal
to that of the cortico-spinal tract—gives a definite movement of the arms.
The focal point in this area—that is, the most excitable point in it—lies
ventral to the central canal. The area includes that of the nucleus ruber
and of the posterior longitudinal bundle.
Stimulation within this area upon one side of the mid-brain is accom-
panied by the assumption of a definite posture on the part of the animal.
The back of the head is twisted towards the same side and the face away
from it, the neck is bent concave to the same side (sometimes the face seems
to be turned to the same side). The arm of the same side is flexed, that of
the opposite side is extended. The lower limb of the same side is extended
and the opposite one flexed (but at one period in an experiment in which this
was usually the case I observed the ipsilateral hind limb to be flexed and
the contralateral to be extended). The tail is bent to the same side. I have
not been able carefully to examine the movements of the trunk.
When stimulation has ceased the posture is maintained. Thus if the
attention be directed to the movements of the arm muscles alone it is found
that the ipsilateral flexion (or contralateral extension) may outlive the
evoking stimulus for several minutes.
1. The Ipsilateral Reaction—When the movements of individual muscles
in the arm are examined (eg. fig. 2) it is found that stimulation of the
ipsilateral area is immediately followed by a sharp flexor contraction. This
soon attains a maximum at which it persists throughout the application of
the stimulus. If extensor tonus is in being the flexor contraction is accom-
panied by reciprocal extensor relaxation. Sometimes during a long applica-
tion of the exciting stimulus an extensor contraction—accompanied by slow
flexor relaxation—may appear late in the period of stimulation. On with-
drawal of the stimulus there is usually no relaxation of the state of flexor
contraction, which then may persist for many minutes. This is the typical
reaction, and by far the most common. But flexor relaxation occasionally
occurs at the termination of stimulation, and this may be followed by an
extensor terminal contraction which is comparatively well maintained. All
these types of reaction have been seen 10 months after the division of all
the posterior spinal roots supplying the left arm.
2. The Contralateral Reaction—The result of stimulation of the contra-
lateral area is to evoke a contraction in the extensor muscle (eg. fig. 4).
Actuities of the Mid-Brain. 149
This is accompanied by reciprocal flexor relaxation if there is flexor tonus at
the commencement of stimulation. The extensor contraction is a more slow
movement than the flexor contraction in the ipsilateral reaction. Having
attained a maximum this persists throughout the period of stimulation and
is continued after termination of stimulation as extensor postural after-
discharge. This is often as well maintained as the flexor after-discharge in
the ipsilateral reaction, but sometimes it dies away more rapidly. Occasion-
ally augmented extensor contraction may be seen, and it sometimes happens
that the terminal phenomena consist of extensor relaxation and flexor
rebound contraction. This is rare and has occurred when there was con-
siderable flexor tonus in being at the time of application of the ipsilateral
stimulus—although even in these circumstances extensor after-discharge is”
the more common. The flexor rebound has been observed to change to
extensor after-discharge after mesial longitudinal section of the mid-brain.
Good after-discharge may be seen in the “ de-afferented ” condition.
3. The Synchronous Compounding of Ipsilateral and Contralateral Reactions.—
The two reactions may obviously be synchronously compounded in such a
manner that the ipsilateral interrupts a contralateral “ background” or the
contralateral an ipsilateral “ background.”
When compounded against an ipsilateral “ background ” (flexion) the effect
of stimulation of the contralateral area (extension) is to produce relaxation
of the “ background ” flexor contraction. This may be complete or it may be
incomplete. When the relaxation is not complete it is found that stronger
contralateral stimulation produces greater flexor relaxation during double
stimulation. The flexor relaxation may be accompanied by reciprocal
extensor contraction—which is, however, not so great in extent as that
in the “ pure” contralateral reaction (fig. 1). On the other hand there may
appear no extensor contraction during double stimulation—even when that
is present in the “pure” contralateral reaction. Although the extensor
contraction is a slow one the flexor relaxation is a very rapid movement, but
the latency of flexor relaxation is usually great. When the interrupting
contralateral stimulus is withdrawn and the ipsilateral stimulus is continued
there occurs a restitution of flexor contraction. This is usually a rapid
movement even where there is a good extensor after-discharge in the contra-
lateral reaction. The restituted flexor contraction may attain a level as
great as that at the corresponding point in a “pure” ipsilateral reaction
(fig 3, reaction “a”). Withdrawal of the ipsilateral “ background” stimulus
is followed by a flexor after-discharge just as in the “pure” reaction. In
one instance an extensor terminal contraction and flexor terminal relaxation
were seen. With the exception of the last phenomenon and of extensor
150 Mr. Graham Brown.
inator
longus.
1 Signal,
Triceps,
QS
Se.
Fig. 1.
HG
152cm™m.,
Fic. 1.—Experiment M, X XIX, record 327, 8860; 1.6.13.—WMacacus rhesus. The record
was obtained 1 hour after decerebration, and 12 minutes after mesial longitudinal
Activities of the Mid-Braan. 151
division of the mid-brain. The letters G—H (ordinates g, g’-h, h’) denote the period
of stimulation of the ipsilateral “dorsal focal point” (posterior longitudinal
bundle 2) in the cross-section of the mid-brain at the anterior colliculi. The letters
K-L (&, #-/, ) in a similar manner denote contralateral stimulation. The upper
record registers contraction (up) and relaxation (down) of the elbow flexor—
supinator longus. The lower record registers similar movements of the elbow
extensor—humeral head of triceps. Below these are the signal lines and a time
tracing which registers seconds. A millimetre scale is reproduced, having been
drawn upon the record before varnishing.
The first reaction is an ipsilateral one. On withdrawal of the stimulus the flexor
after-discharge is extremely poor.
The second reaction is a contralateral one. Extensor contraction occurs. This is
here accompanied by abnormal flexor contraction—not usually seen. There is an
extensor after-discharge which is not well marked, but a sudden relaxation of this
is seen at the commencement of the third reaction, which opens with the ipsilateral
reaction.
In the third reaction the two stimuli are compounded synchronously with an
ipsilateral “background.” In the phase of double stimulation (4, #-1, /’) there is
extensor contraction and a partial and slight flexor relaxation. In this phase small
rhythmic irregularities are evident in the two records. These are related to a
slowing and deepening of respiration which then occurred. It will be observed that
the extensor contraction is less in extent than that of the “pure” contralateral
reaction (the second reaction of the record). On withdrawal of the contralateral
stimulus there occurs flexor restitution and extensor relaxation. Flexor after-
discharge occurs on withdrawal of the ipsilateral “ background ” stimulus, and is
much better sustained than that in the preceding “ pure” ipsilateral reaction.
In the fourth reaction of the record the “ background ” is contralateral (extension).
The contralateral stimulus is applied in the flexor after-discharge of the preceding
reaction and causes flexor relaxation (at &, k’). During double stimulation (g, g’-
h, h’) there occurs a partial and slight extensor relaxation accompanied by reciprocal
flexor contraction. Withdrawal of the ipsilateral stimulus is followed by flexor
relaxation and extensor restitution of contraction. Withdrawal of the “ back-
ground ” contralateral stimulus is followed by extensor after-discharge.
This figure demonstrates the presence of flexor after-discharge in the ipsilateral
reaction and extensor after-discharge in the contralateral ; of the effects of com-
pounding the two in temporal succession; and of the effects of synchronous
compounding with ipsilateral and contralateral ‘‘ backgrounds,” all after mesial
longitudinal division of the mid-brain.
contraction during double stimulation all these points have been observed in
the “ de-afferented ” condition as well as in the “normal.”
When compounded against a contralateral “ background” (extension) an
interrupting ipsilateral stimulus (flexion) evokes extensor relaxation and
flexor contraction. Where the ipsilateral stimulus is comparatively weak
the extensor relaxation may be incomplete (fig. 2). Where stronger it may
be complete. Withdrawal of the interrupting ipsilateral stimulus is accom-
panied by a sharp relaxation of the flexor contraction. This may occur even
when there is a flexor after-discharge in the ipsilateral reaction and when
the contralateral “ background” stimulus is ineffective. The flexor relaxation
152 Mr. Graham Brown.
when the contralateral stimulus is effective may yet not be accompanied by
restitution of extensor contraction. But that restitution may occur (figs. 1, 2).
It then is a slow movement and closely resembles the extensor contraction
in the “pure” contralateral reaction. Sometimes the flexor relaxation on
withdrawal of the interrupting ipsilateral stimulus is a slow one (figs. 1, 2).
Occasionally the flexor contraction may even be sustained after withdrawal
of the ipsilateral stimulus (fig. 3). In such cases the flexor contraction is
Supinator
x -
longus
Trice ps.
i Szgnal.
Signal.
(YY
Wecondls:
Fic. 2.—Experiment M, X XIX, record 327, 8855; 1.6.13.—d/acacus rhesus. From the
same experiment as fig. 1. This record was obtained 42 minutes after decerebration
and before the'mesial longitudinal division of the mid-brain.
The first reaction is here an ipsilateral one, and it is applied during the presence
of an extensor tonus from a preceding contralateral reaction. On stimulation
extensor relaxation and flexor contraction occur. The ipsilateral reaction is
followed by a good flexor after-discharge.
The second reaction is a contralateral one. Here there occur flexor relaxation
and extensor contraction. The extensor contraction is again rhythmically notched.
The extensor after-discharge is not well marked.
The third reaction is compound. The contralateral stimulus is first applied, and
is then interrupted by an ipsilateral. During double stimulation (g, g’-/, h’) there
is flexor contraction and extensor relaxation. The latter is not to so low a level,
and the former is not to so high a level as those in the “pure” ipsilateral reaction.
Withdrawal of the ipsilateral stimulus is followed by a slow flexor relaxation (it is
usually more rapid) and by extensor restitution of contraction.
Compare this figure with fig. 1 (after mesial longitudinal division of the mid-
brain). Here the effects of compounding the two reactions synchronously and in.
temporal succession are demonstrated as they occurred before the lesion.
Actiwities of the Mid-Brain. 153
not usually sustained on withdrawal of the contralateral “background”
stimulus. Where extensor restitution of contraction occurs the withdrawal
of the contralateral stimulus is followed by extensor after-discharge. In one
Supinator
longus.
Con. .
fe 75 CU - +f
Gl 15 Fe aa
Signal.
Seconds,
a. b.
Fic. 3.—Experiment M, XXIV, record 311, 8362 ; 31.3.13.—Macacus rhesus. The dorsal
spinal roots of the left (recording) fore limb divided in May, 1912. A reaction
obtained 44 minutes after decerebration.
The first reaction (a) is a compound one with an ipsilateral “ background.”
During double stimulation there is flexor relaxation, but no extensor contraction.
The latency of the flexor relaxation is great. On withdrawal of the contralateral
stimulus there is flexor restitution of contraction which occurs as a very sudden
movement. On withdrawal of the ipsilateral “background” stimulus there is a
marked flexor after-discharge. The sudden drop in the flexor after-discharge seen
about 3 mm. before the final ordinate in reaction (a) occurred during a period of
15 seconds in which the kymograph was stopped—it therefore represents a very
slow movement.
The second reaction (6) is a compound one in which the contralateral reaction is
the “background.” During double stimulation flexor contraction and extensor
relaxation occur. But on withdrawal of the ipsilateral interrupting stimulus (at
h, h’) there continues a flexor after-discharge. On withdrawal of the contralateral
“background ” stimulus this disappears.
case, where flexor relaxation occurred on withdrawal of the interrupting
ipsilatera] stimulus, there yet occurred flexor rebound on withdrawal of the
contralateral “ background” stimulus. There the same phenomenon occurred
VOL. LXXXVIL—B. M
154 Mr. Graham Brown.
in the “ pure ” contralateral reaction. With regard to the phenomena described
in this paragraph there is little difference between the “ de-afferented ” and
the “normal ” conditions.
It therefore appears that the phenomena obtained when the two areas in
the cross-section of the mid-brain are simultaneously stimulated closely
resemble those obtained when the movements of two antagonists in response
to peripheral nerve stimulation are examined. “Algebraic summation”
seems to occur, and the phenomena which occur when the interrupting
stimulus is withdrawn and the “ background ” still continued nearly approxi-
mate to those seen under similar conditions in the peripheral reflexes where
the “pure ” reactions are followed by good “ after-discharge.”
4. The Compounding of Ipsilateral and Contralateral Reactions in Temporal
Succession— Where the ipsilateral reaction is followed by flexor after-
discharge and the contralateral by extensor after-discharge the two reactions
may be compounded in such a manner that the one falls during the after-
discharge of the other. If this is done the contralateral reaction (extension)
at once produces a very sharp relaxation of the flexor after-discharge of the
ipsilateral reaction, On withdrawal of the contralateral stimulus an extensor
after-discharge is left in being, and this is at once reduced if the ipsilateral
stimulus is repeated, and so on (fig. 2). If the contralateral stimulus is weak
or of very short duration there may be incomplete relaxation of a flexor after-
discharge. If the ipsilateral stimulus is weak there may be a partial relaxa-
tion of an extensor after-discharge, and this may thereafter be reconstituted.
5. The Geographical Position of the “ Focal Point.,—When stimulation
applied to one or other side of the cross-section of the mid-brain gives one
or other of these reactions it is usually found that the area from which they
may be obtained is comparatively large. The minimal reaction may, how-
ever, be localised to a comparatively small area. This area (“ focal point”)
lies about 3 to 4 mm. ventral to the dorsal surface of the mid-brain, and
about 2 to 3 mm. from the mesial plane. The surrounding parts are
inexcitable, except, perhaps, those immediately between the focal point and
the mesial plane.
In one experiment in which decerebration was comparatively high—the
division of the neuraxis passing just oral to the anterior colliculi—the focal
point was found to be much more ventral than this, about 7-8 mm. from the
dorsal surface. The ipsilateral reaction was of the usual type. ‘The contra-
lateral reaction gave relaxation of a flexor after-discharge (if that was in
being) and extensor contraction, but on withdrawing the stimulus there was
at once sharp extensor relaxation and a marked flexor rebound contraction.
The mid-brain was then split in the mesial plane (the section was found to
Activities of the Mid-Brain. 155
have passed out of the mesial plane into the left half of the neuraxis at
the level of the posterior colliculi). Immediately thereafter the focal point
was found to be in the usual more dorsal position. Ipsilateral stimulation
gave the usual reaction, and contralateral stimulation gave the same reaction
as before, save that there was marked extensor after discharge and no flexor
rebound contraction.
V. The Effect of Various Lesions.
1, Mesial Section between the Right and Left Halves of the Mid-brain.—
When the reactions are obtained from the dorsal focal point there may be no
change in them after this lesion. The phenomena during the immediate and
successive compounding of the two reactions may be the same as before
(figs. 1, 4). The excitability may be depressed slightly, or it may remain
unchanged, or it may even appear to be raised. The effects of mesial section
in a case where the lower focal point was effective have been described in
the previous section.
2. Right Semi-section of the Mid-brain between Anterior and Posterior
Colliculi.—After this-lesion it is found that the ipsilateral reaction (from the
left side of the mid-brain) is unimpaired, but the contralateral reaction (from
the right side of the mid-brain above the level of the semi-section) is
abolished. The contralateral reaction may at once be obtained by stimulation
of the caudal surface of the cut in the mid-brain.
3. Division of the Right Superior Cerebellar Peduncle-—This was found to
have no appreciable effect upon the reactions and their compound effects.
The experiments upon the cerebellar peduncles, in view of the effects of
removal of the whole cerebellum, were not continued.
4, Complete Removal of the Cerebellum—lIn several experiments the
cerebellum has been completely removed. After this lesion there is at first
no change in the two reactions. Flexor after-discharge follows the ipsilateral
reaction and extensor after-discharge the contralateral, and the effects of
compounding the two in temporal succession is the usual one (fig. 5). This
may last for 30 minutes or more. Thereafter the flexor after-discharge
disappears, the withdrawal of the ipsilateral stimulus being followed by sharp
flexor relaxation. In one experiment the extensor after-discharge still
persisted. No change in the excitability of the reactions may occur. In
other cases the flexor after-discharge may disappear from the moment of
removal of the cerebellum (the reactions have usually been tested within one
minute of that removal). In one case the contralateral reaction reversed to
flexion with the same strength of stimulus which before the removal gave
extension. The procedure of removal of the cerebellum has been observed
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5. Removal of Mid-brain.
discharge in the ipsilateral reaction outlasted for some time the removal of
the cerebellum it was found that it also outlasted removal of part of the
was present immediately after the removal.
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The effect of heat upon a peptone solution is thus exhibited :—
solution.
Fibrinogen
Thrombokinase
(5-per-cent.
solution
of peptone).
0 ‘85-per-cent. DeSes DEse eae
ecocoooscoooSos
WHONYNNYNNNYNNHYHYNY®
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Coagulation
time (30° C.).
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TISBOND NEON
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(©) S\(o) (Se {=] (=)
Sl eg ee le
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co w co co Ww
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90 minutes.
15 5
3 hours.
* Previously heated to 60° for 5 minutes.
With the addition of increasing amounts of peptone in presence of
calcium chloride delay of the onset of coagulation was observed, the coagula
being very soft.
The Action of Viper Venom upon Circulating blood Plasma.
The lethal amount of the venom of Hchis carinatus (administered by intra-
venous injection) was found by C. J. Martin to be for the rabbit about
half a milligramme per kilogramme of body weight ; if a large dose of viper
venom was injected rapidly into a vein, intravascular clotting occurred ;
if a relatively small dose was injected very slowly, the coagulability of the
Coagulant of the Venom of Kchis carinatus. 187
blood was diminished. It may here be observed that very small doses
(0:03 mgrm. or less per kilogramme of body weight) fail to produce any
recognisable ill effect in rabbits.
The effect of intravenous injection of viper venom upon circulating blood
plasma was studied in a series of experiments similar to the following :—
Experiment 4.—A rabbit weighing 384 grm. received into the vein of the
ear an injection of 2 c.c. of 0°65-per-cent. sodium chloride solution containing
0:13 megrm. of viper venom (0°34 mgrm. per kilogramme of body weight).
Five minutes after the completion of injection, which occupied one minute,
the animal, which had not up to this time been obviously affected, showed
sions of feebleness, and then became convulsed, death occurring about three-
quarters of a minute later. At the post-mortem examination, which was
made without delay, the blood in the superior vena cava, the right auricle
and ventricle of the heart and the pulmonary artery was found to be
clotted, the clot extending into the vein injected; on the left side of the
heart, in the aorta, portal vein, and lower part of the vena cava, liquid
blood was found; the lungs collapsed normally on opening the chest.
On microscopical examination of the lungs after death by viper venom it
was found that in all cases, no matter whether intravascular coagulation
resulted (four experiments) or the blood coagulated slowly (three experi-
ments) or remained permanently liquid (three experiments), fibrin masses,
filaments, and fibrils could be readily recognised. The appearance presented
by the collections of fibrin was exactly the same as that presented when
relatively large doses of thrombin solution had been injected, so that it was
not possible from an examination of the lungs to state whether the coagulant
injected had been the latter or the former. The diminished coagulability or
“negative phase” exhibited after the intravenous injection of viper venom
is therefore dependent upon the removal of fibrinogen under the action of
the coagulant. As already mentioned, Martin* observed that the slow
injection in the dog of small quantities (7.2. below 0°71 mgrm. per kilo-
gramme of body weight) of viper (Pseudechis porphyriacus) venom resulted
in the production of liquid blood, and it is easy to understand how this
procedure, by allowing time for the contraction of the delicate fibrin
network, which is at first formed, into dense masses of relatively small size,
would cause far less blocking of vessels and resulting interference with the
circulation than would the rapid injection of the same or smaller amounts
of venom. Nevertheless, in my experiments upon rabbits, liquid blood has
followed the injection (in the course of about one minute) of relatively large
* C. J. Martin, 1893, Joe, cit., p. 382.
188 Dr. J. O. W. Barratt. The Nature of the
quantities of venom (34-0°6 megrm. per kilogramme of body weight).
Liquid blood, moreover, does not exclude the presence of visible clot in the
larger vessels. Thus in the experiment referred to at the close of the next
section, in which 0°73 mgrm. of venom per kilogramme of body weight was
injected into a rabbit, it was found that the heart was continuing to beat
feebly and slowly after respiration had stopped and all movements of the
voluntary muscles had ceased. On opening the right ventricle 5 e.c. of
blood was obtained, which remained permanently liquid. The vein of the
ear, external jugular, superior and inferior caval veins, portal and renal
veins were all distended with clot; nevertheless, obstruction to the circula-
tion was not complete, and blood was delivered from the incised right
ventricle at each beat. In the blood-vessels of the lungs fibrin filaments and
masses were abundant.
That the appearance of liquid blood after injection of viper venom is
dependent upon removal of fibrinogen is shown not only by the appearance
of fibrin in blood-vessels, particularly of the lungs, but also by the result
of examination of the fibrinogen content of the liquid blood. Thus it was
found that when one part of the thrombin solution employed in the pre-
ceding section was mixed with two parts of the liquid blood obtained im the
experiment just described, coagulation did not occur, though the thrombin
solution was capable of coagulating two parts of fibrinogen solution in about
three-quarters of a minute. The liquid blood was, however, found to
contain thrombin, for when added to two parts of fibrinogen solution
coagulation occurred at the end of one and a half minutes; the same result
was obtained if one-fifteenth part of 0°83-per-cent. potassium oxalate
solution had also been previously added. The thrombin in question cannot
have been produced by the action of venom upon fibrinogen, for serum
expressed from a coagulum so obtained, as, for example, in the second
experiment of the series given on p. 189, exhibits the usual character of serum
from a clot produced by the action of thrombin upon fibrinogen solution
under similar conditions of experiment, that is to say, it possesses scarcely
any recognisable coagulant action on fibrinogen. It follows, therefore, that
the thrombin contained in the liquid blood consists of venom. In the
experiment in question the amount of venom injected into the blood
represented 1 part in 70,000 parts of blood. In the next section it is shown
that 1 part in 15,000,000 is capable of coagulating a solution containing
fibrinogen in approximately two-fifths of the concentration present in blood
plasma ; it is therefore to be expected that part of the venom injected would
remain unchanged in the liquid blood, thereby conferring upon it a coagulant
action when added to fibrinogen solution.
Coagulant of the Venom of Wchis carinatus. 189
Even when, after the injection of a lethal dose of viper venom, clotting
oceurs in the heart and great blood-vessels, the ease with which fibrin masses
can be recognised in the smaller pulmonary vessels renders it possible
readily to distinguish between the effect of injection of thrombin or viper
venom and that of a relatively large amount of thrombokinase contained in
red-cell stromata or peptone. It is obvious from the effect of injection of
viper venom into the blood stream that the coagulant in the venom is a
thrombin and not a thrombokinase.
The Effect of Heat wpon Viper Venom.
The blood coagulant of the venom of the Indian viper, Hchis carinatus, is
completely destroyed by heating to 75° C. for 10 to 15 minutes.* The
following experiments exhibit the coagulant activity of this venom before
and after heating :—
Solution of Orsauper cont
Rabaowen'| viper venom 0 '6-per-cent. | 0 °85-per-cent. seagate ‘ GowmilaGon
age es 1 in 300,000 of CaCl, NaCl Rarer CAN: ae 30°C.)
eee 0 *85-per-cent. solution. solution. Ree Hue sue
oxalate.
NaCl.
|
¢.c Gc Cc: C.c. (Ooh,
0:2 — 0:03 0°27 — 90 minutes.
0°2 0-01 0°03 0°26 — 10 Me
0-2 0:03 0:03 0-24 — 5 a
0-2 0-10 0-03 0°17 — 4 a
0:2 0°30 0:08 — oo eens es
0-2 0 :30* 0:05 — — 53-60 __,,
0-2 — — 0°27 0°03 3 hours |
0-2 0-01 — 0°26 0-03 16 minutes.
0-2 0°03 — 0°24 0-03 10 .
0-2 0:10 — 0°17 0-03 7
0°2 0°30 — a 0-03 4 S
0-2 | 0 *380* — = | 0-03 60 Re |
* Heated to 75° for 10 minutes.
It will be seen that viper venom, even when present to the extent of only
1 part in 15,000,000, is capable of coagulating in ten minutes at 30° C. a
liquid containing approximately the same concentration of fibrinogen as.
normal rabbit’s plasma. When heated, however, its coagulant action rapidly
disappears.
It is obvious from the effect of heating that the coagulant of viper venom
cannot be regarded as thrombokinase as Mellanbyft has suggested. Its
behaviour in respect of heat shows it to be a thrombin.
* C. J. Martin, 1905, Joe. cit.
+ J. Mellanby, Joc. cit., p. 467.
VOL. LXXXVII.—B. P 1A.
190 Mr. Porter and Dr. Edridge-Green. Negative After-Images
The intravenous injection of heated venom, even in the amount of
1:1 merm. per kilogramme of body weight, was found to be without effect
upon rabbits; examination of sections of the lungs and also of the liver,
spleen, kidney, and heart muscle of animals killed half an hour after
injection failed to reveal any separation of fibrin in the blood-vessels. A
control experiment, in which 0°73 mgrm. per kilogramme of body weight was
injected in the course of one minute, caused death at the end of 30 seconds.
The behaviour of heated venom, when injected into the blood stream, is
thus seen to be consistent with the conclusion that the coagulant is a
thrombin.
Summary.
The different mode of action exerted by thrombin and thrombokinase upon
circulating blood plasma is described, and it is shown that the coagulant of
viper (Hchis carinatus) venom, as exhibited by its effect in causing intra-
vascular separation of fibrin when injected into the blood stream, and also
indicated by its behaviour when heated, is a thrombin and nota
throm bokinase.
Negative After-Images and Successive Contrast with Pure
Spectral Colours.
By A. W. Porter, B.Sc., F.R.S., Fellow of University of London University
College, and F. W. Eprincr-Green, M.D., F.R.C.S.
(Received March 31,—Read November 13, 1913.)
In a recent paper* Prof. Burch has criticised our results on “ Negative
After-Images and Successive Contrast with Pure Spectral Colours.”
Prof. Burch suggests that the change in blue and violet obtained after
fatigue with red light may be explained on the Young theory, if the stray
light, which we stated was present, be taken into consideration. He states
that the reason, on this theory, why the violet appeared bluer and darker
after fatigue to red was due to the elimination of the red component in
the stray light.
In consequence of this criticism we have since repeated our experiments,
taking the most minute precautions to exclude stray light by covering the
* ‘Roy. Soc. Proc.,’ 1913, B, vol. 86, p. 117.
+ ‘Roy. Soc. Proc.,’ 1912, B, vol. 85, p. 434.
and Successive Contrast with Pure Spectral Colours. 191
whole apparatus and head of the observer with black velvet. When these
most minute precautions were taken to prevent the admixture of red or
other light the results were exactly the same as before. The experiments
were conducted as follows: A region of pure violet, \ 4368-A 4572, was
isolated in the Edridge-Green spectrometer, a deep blue-green glass quite
opaque to red being placed in front of the slit, so that no red light could
enter the instrument. A region of pure red, \ 6360-6570, was isolated
in another spectrometer, deep ruby glass being placed in front of the slit so
that nothing but red light could enter the instrument. The eye was then
fatigued as before, one eye being vertically above the other, for 20 seconds,
and the after-image projected upon a narrow vertical band in the violet
region after turning the eyes round into the normal position, so that the
two images crossed at right angles. The result was exactly the same as
stated previously by us, the region of violet crossed by the after-image
appeared bluer and darker.
It should be here noted that when the red band was intently regarded for
10 seconds and the eye then slightly moved (to another part of the same
telescopic field) a bright blue-green after-image was visible, although the only
light then being received by the eye was red light.
The experiment with yellow light on a screen was repeated in the spectro-
meter with exactly the same result. Pure yellow light, \ 5820-X 5870, was
isolated in one spectrometer, and red light, \ 6360-2 6570, used to fatigue
the eye. The results were as before, the yellow appeared unchanged, or,
when the exciting light was comparatively intense, slightly greener and
darker in the region of the after-image, whilst a deep blue-green after-
image extended on either side.
These experiments show that the stray light, mentioned by us in our former
paper, was of negligible amount; for we have now obtained precisely the same
results when stray light was most rigorously excluded. Stray light, of amount
comparable with that in our previous experiments, is present in all spectro-
metric investigations unless precautions such as those described above are
taken.
192
The Ratio between Spindle Lengths in the Spermatocyte Meta-
phases of Helix pomatia.
By C. F. U. MmEk, M'Sc., E.LS., F.Z.8.
(Communicated by Sir W. T. Thiselton Dyer, K.C.M.G., C.1E., F.R.S. Received
July 15,—Read December 4, 1913.)
[PuatE 12.]
Introduction.
I have recently shown that in Forjficula auricularia the length of the
mitotic spindle, we. the distance between the centrosomes, seems to be
a constant at the conclusion of each spermatocyte metaphase. The ratio
between the lengths found at this stage is almost identical with the ratio
between the radii of two spheres of which the volume of one is equal to
twice that of the other; and, since the volume of the primary spermatocyte
cell in the metaphase is presumably equal to twice that of the secondary
spermatocyte, connection is suggested between the length of the spindle and
the volume of the cell.
I now propose to measure spindle lengths in the spermatocyte metaphases
of Helix pomatia. As in the case of Forficula, the chromosomes are spheres
or very short rods, and all seem to divide on the spindle at the same time ;
the conclusion of each metaphase is therefore easily recognised. If the
lengths are found to be constants, and if the ratio between them is
approximately 1:26:1, the connection between spindle length and cell
volume is again suggested: if, on the other hand, lengths are not constants,
or if the ratio between them is not approximately that mentioned above, the
suggested connection is at once disproved.
Material and Methods.
The material, which consisted of the hermaphrodite gland, was obtained at
the end of May, and was preserved in Flemming’s strong chromo-aceto-osmic
acid fluid and the platino-aceto-osmic acid fluid of Hermann. The material
remained in the fixative for i2 hours, and, after being washed thoroughly in
running water and passed through successive strengths of alcohol, was
embedded in paraffin. Sections were cut 8 thick with an ordinary
Cambridge rocking microtome.
The stains used were Heidenhain’s iron hematoxylin and iron brazilin,*
* Hickson, 8. J., ‘Quart. Journ. Micro. Sci.,’ 1901, vol. 44.
Spindle Lengths in Spermatocyte Metaphases of Helix pomatia. 193
and both have given excellent results. The latter, which affects the
cytoplasm as well as the chromatin, enables spindle fibres to be seen very
distinctly ; and all drawings on the plate have accordingly been made from
sections thus stained. In the case of the iron hematoxylin, the slides were
placed for four hours in the mordant, which was an aqueous solution of
ferric alum, and were then stained for 12 hours; in the case of the iron
brazilin, the slides remained for two hours in a solution of ferric alum in
70-per-cent. alcohol, and were then placed in the stain for 15 hours.
The preparations were studied with a Zeiss apochromatic oil-immersion
objective of 2 mm. focus and N.A. 1°30, and compensating oculars Nos. 4, 6,
12, and 18. The light was obtained from an inverted incandescent gas
burner, and was passed through a Gifford screen and the holoscopic oil-
immersion substage condenser of Messrs. Watson and Sons, of London. All
drawings were made with a large Abbé camera lucida at one magnification,
which was estimated with a stage micrometer graduated to read one-
hundredth part of a millimetre. Possible distortion was prevented by
levelling the microscope platform and drawing table; and, in order to
minimize error due to foreshortening, measurements have been made only of
spindles of which the major axes lay at right angles to the microscopic line of
vision, 7.¢. spindles of which the centrosomes could be brought into focus
simultaneously. I have tried to eliminate inaccuracy of draughtsmanship
by drawing the centrosomes of each spindle many times and upon several
occasions; moreover, the lengths found by me have been checked by
independent measurements made by my assistant, Mr. Russell Goddard.
The Length of the Mitotic Spindle at the Conclusion of the Primary
Spermatocyte Metaphase.
Fig. 1 of the plate represents a polar view of the primary spermatocyte
complex. I have not attempted to count the chromosomes on the various
spindles; but recent investigations seem to show that the number is 48 in
the spermatogonial and 24 in the spermatocyte cells. The chromosomes are
short thick rods, and do not differ from one another greatly in size.
Figs. 2 to 12 inclusive are drawings of lateral views of the spindle at the
conclusion of this metaphase; in each figure constriction of the chromosomes
is seen to have been completed, and the daughter rods, apposed to one
another in the equatorial plane, are ready to move apart. These drawings
have been made at a magnification of 650 diameters from sections in the
hermaphrodite glands of several individuals, and the length of the spindle is
the same in all. This length has been found in every primary spermatocyte
cell studied at this stage, and, at the known magnification, represents 15:3 p.
194 Mr. C. F. U. Meek. The Ratio between Spindle
In the circumstances we have reason for believing that the length of the
spindle is a constant at the conclusion of the primary spermatocyte metaphase.
The Length of the Mitotic Spindle at the Conclusion of the Secondary
Spermatocyte Metaphase.
. Fig. 13 represents a polar view of the equatorial plate in this metaphase.
The chromosomes are noticeably smaller than those of the preceding cell
generation.
Figs. 14 to 23 inclusive are drawings of lateral views of the spindle at
the conclusion of the metaphase, 7.e. at the moment when constriction of
the chromosomes is complete. As in the case of figs. 2-12, these drawings
have been made at a magnification of 650 diameters from sections in the
hermaphrodite glands of several specimens. The length of the spindle,
estimated from the magnification, is invariably 12:1 ~; and, since the centro-
somes have been found to be equidistant in all secondary spermatocyte cells
studied at this stage, we seem again to be dealing with a constant.
The Ratio between the Lengths of the Mitotic Spindle at the Conclusion of the
Primary and Secondary Spermatocyte Metaphases.
I have already remarked that in Forficula auricularia the ratio between
the lengths of the mitotic spindle at the conclusion of the twe spermatocyte
metaphases is almost identical with the ratio between the radu of two
spheres of which the volume of one is equal to twice that of the other. The
former ratio is 1°28: 1°00, and the latter ratio is 1°26: 1:00.
Now the lengths of the spindle found for the conclusion of these meta-
phases in Helix pomatia are 15:3 and 12:1 respectively, and the ratio
between them is 1:26:1:00. No period of growth separates the primary and
secondary spermatocyte mitoses in this organism; the connection between
spindle length and cell volume is therefore again suggested.
The accuracy of my measurements seems to be confirmed by the work of
Demoll. In a paper published last year upon the spermatogenesis of Helix
pomatia, he gives two drawings respectively representing the lengths of the
mitotic spindle in the primary and secondary spermatocyte metaphases. We
are not told at what stage of the metaphase these drawings were made, nor
is the magnification mentioned ; but the lengths shown are 29-7 and 24:0 mm.,
and the ratio between them is 1°24:1:00. Demoll, however, appears to have
seen no possible significance in these relative lengths; for he dismissed
the matter by saying that the length of the spindle decreases only slightly
when the cell volume is halved.
Lengths in the Spermatocyte Metaphases of Helix pomatia. 195
Measurements of Spindle Lengths made by an Independent Investigator.
During this research I wrote upon the subject to Dr. von Winiwarter,
who very kindly offered to measure spindle lengths in the spermatocyte
metaphases of man.
I have since received a letter, in which he says: “J'ai effeetué une série
de mensurations sur les fuseaux des spermatocytes I et II chez homme, et
constaté quil y a une légére différence de longueur entre les deux. Cette
différence est trop faible pour étre reconnue autrement que par des
mensurations. Je vous envoie en méme temps quelques uns des croquis
qui m’ont servi a calculer les rapports. Ils sont faits 4 un grossissement de
2400 diamétres avec le systtme docul. et dobject. employé pour tous les.
dessins de mon travail sur la spermatogenese humaine (‘ Arch. de Biol.’).
J’ai simplement indiqué les corpuscles centraux, le début du fuseau et les
contours du corps cellulaire. Je n’ai pas dessiné les chromosomes ; ceux-ci
sont exactement au moment ou ils sont rangés régulicrement a l’équateur et
vont se diviser. Les fuseaux sont bien paralleles a la table du microscope
et dans une seule coupe. Vous constaterez vous méme que l’analogie avec
forficula est tellement complete que le rapport entre les fuseaux I et IT est
aussi 1:26 : 1:00.”
The camera lucida drawings enclosed in this letter represent five primary
and five secondary spermatocyte metaphases. The length of the spindle is
24 mm. in each of the former, and 19 mm. in the latter; and, since the
magnification is 2400 diameters, the lengths in the cell must be 10:0 and
79 w respectively. Examples of these drawings are given below at a slightly
A. B.
Camera lucida drawings of spindles in the spermatocyte metaphases of Man.
A. Primary spermatocyte. B, Secondary spermatocyte. x 2270.
reduced magnification. In the circumstances the ratio that I have observed
in Porficula auricularia and Helix pomatia is shown to exist in material
belonging to a third phylum; and I take this opportunity of again thanking
Dr. von Winiwarter for his kindness in making the measurements and
allowing me to publish the results. *
196 Mr. C. F. U. Meek. The Ratio between Spindle
Conclusion.
Whether the connection suggested between spindle length and cell volume
in the metaphase is likely to be established or not is impossible for us to
say: the proposition that I have put forward is at present entirely specu-
lative. But the results of research have shown that at the stage in question
the ratio between spindle lengths is approximately the same in the spermato-
cytes of Helix pomatia, Forficula auricularia,and man—organisms representing
three phyla of the animal kingdom.
Moreover, consideration of the lengths found in these organisms proves
that the length of the spindle in the metaphase cannot be correlated with
the volume of the chromatin. This is important; for in an earlier paper I
have produced evidence to show that increasing somatic complexity is
accompanied by increase of chromatin volume in the cell.
The failure of current theories of mitosis is largely due to the absence of
data from which to draw conclusions; and, since either proof or disproof
of my proposition must constitute a new generalisation, I intend to carry
out further and similar cytometrical investigations, of which the results will
appear in subsequent papers.
Summary.
1. The length of the mitotic spindle, zc. the distance between the centro-
somes, is 15°3 w at the conclusion of each primary spermatocyte metaphase
of Helix pomatia.
2. The length of the mitotic spindle is 121 at the conclusion of each
secondary spermatocyte metaphase of Helix pomatia.
3. The ratio between the lengths of the mitotic spindle at the conclusion
of the primary and secondary spermatocyte metaphases is approximately
the same in Helix poimatia, Forficula auricularia, and man; and, since these
ratios are either identical or almost identical with the ratio between the
vadii of two spheres of which the relative volumes are the same as those of
the cells in question, connection may exist between spindle length and cell
volume at this stage.
4. A comparison of mitotic figures in Helix pomatia, Forficula auricularia,
and man proves that the length of the spindle in spermatocyte metaphases
cannot be correlated with the volume of chromatin in the cell.
Meek. Roy. Soc. Proc., B, vol. 87, Plate 12.
21. 22. ZS. ZF.
Lengths in the Spermatocyte Metaphases of Helix pomatia. 197
BIBLIOGRAPHY.
Demoll, R., 1912. “Die Spermatogenese von Helix pomatia, .,” ‘Zool. Jahrb.,’
Supplement XV, vol. 2.*
Meek, C. F. U., 1912. “A Metrical Analysis of Chromosome Complexes, showing Corre-
lation of Evolutionary Development and Chromatin Thread-width throughout
the Animal Kingdom,” ‘ Phil. Trans.,’ B, vol. 203.
Meek, C. F. U., 1912. “The Correlation of Somatic Characters and Chromatin Rod-
lengths, being a Further Study of Chromosome Dimensions,” ‘Journ. Linn. Soc.
Zool.,’ vol. 32.
Meek, C. F. U., 1913. “The Problem of Mitosis,” ‘Quart. Journ. Micro. Sci.,’ vol. 58,
Part IV. F
Meek, C. F. U., 1913. “The Metaphase Spindle in the Spermatogenetic Mitosis of
Forficula auricularia,” ibid., vol. 59, Part IL.
Winiwarter, H. von, 1912. “ Etudes sur la Spermatogenise humaine,” ‘ Arch. de Biol.,’
vol. 27.
EXPLANATION OF THE PLATE.
Fig. 1.—Polar view of primary spermatocyte complex. ‘
Figs. 2-12.—Lateral views of spindle at conclusion of the primary spermatocyte meta-
phase, showing completed constriction of chromosomes. In each figure the
length of the spindle, estimated from the magnification, is 15°3 p.
Fig. 13.—Polar view of secondary spermatocyte complex.
Figs. 14-23.—Lateral views of spindle at conclusion of the secondary spermatocyte meta-
phase, showing completed constriction of chromosomes. In each figure the
length of the spindle, estimated from the magnification, is 12°1 p.
Fig. 24.—Divisions of stage micrometer, 10» apart, showing magnification of figs, 1-23
inclusive.
* A list of publications dealing with the spermatogenesis of Helix is given in the
bibliography of this paper.
198
Neuro-Muscular Structures in the Heart.
By A. F. Sranuey Kent, M.A. Oxon., Professor of Physiology, University of
Bristol.
(Communicated by Prof. C. S. Sherrington, F.R.S. Received July 25,—
Read November 20, 1913.)
(From the Physiological Laboratory of the University of Bristol.)
The fundamental fact of the existence of a muscular connection between
auricle and ventricle in the mammalian heart was established in 1892 (5, 6).
The details of the particular connection first studied were worked out during
the years following (1, 3, 7, 8,13, 15), and attention was directed so com-
pletely to the auriculo-ventricular bundle itself that additional ties between
auricle and ventricle at other points remained relatively neglected. Partly
in result of this, though partly in result of experiments which have been
perhaps imperfectly understood, an impression has gained ground (2, 14) that
apart from the originally described auriculo-ventricular bundle there exists
no other conducting path capable of transferring the state of activity from
auricular muscle to ventricular, or vice versd. This impression has, indeed,
been put forward as an actually ascertained fact (10).
For some years, however, a mass of facts has been accumulating difficult
to explain on the supposition that the conduction between auricle and
ventricle consists of one single path alone. The facts can, on the other
hand, be explained satisfactorily if there be granted the existence of an
auriculo-ventricular connection which is multiple.
These facts have become known partly as the result of clinical experiences
and partly as the result of direct experiment, and are so definite that it is
necessary for any satisfactory theory of the cardiac mechanism to take
account of them.
The clinical experiences referred to fall into two categories :—
A. Cases in which the auriculo-ventricular sequence was found to be
normal, though the bundle was destroyed (4,11); and
B. Cases in which the auriculo-ventricular sequence was abolished, though
the bundle was intact (4, 9, 12).
There are in the literature several cases illustrating each of these condi-
tions, and the conclusion is becoming more and more firmly established, that
the normal auriculo-ventricular sequence may exist with a destroyed bundle,
and that the sequence may be disturbed, or abolished, the bundle remaining
unaffected.
Neuro-Muscular Structures in the Heart. 199
In other words, it appears that the auriculo-ventricular bundle is not the
only path by which the functional connection between the auricle and
ventricle may be established, and that “the co-operation between the auricle
and ventricle is not necessarily dissolved because the auriculo-ventricular
bundle has been put out of action ” (4).
The experimental evidence to which reference has been made is at present
unpublished, and was first brought to my notice by Prof. Leonard Hill, to
whom I am indebted for permission to refer toit. I have recently repeated
the experiments, and can have no doubt as to their real significance.
The evidence is of the following character :—
If in the beating heart of a mammal the anatomical connections between
the left auricle and left ventricle are severed, and the section is carried
through the septum also, thus leaving only the right ventricular wall attached
to the auricle, even under these circumstances co-ordinated beats pass over
the auriculo-ventricular junction, the ventricular contraction following the
auricular in its proper sequence.
With such clinical and experimental evidence before us it is idle to assert
that no conducting path exists other than the well-known and well-defined
auriculo-ventricular bundle, and the question is no longer “ Does a connec-
tion exist ?” but “ What is the nature of the connection ?”
It may perhaps be recollected that as long ago as 1892 (5, 6) I described
the existence of a connection in this situation, viz., between the outer wall
of auricle and ventricle. The importance of the recently-described septal
connections overshadowed this other observation, however, and its significance
was not appreciated. It was only when the fact that the auriculo-
ventricular bundle could be destroyed without abolishing the co-ordinated
action between the chambers that the importance of additional conducting
paths was brought into prominence.
During the past few years my work on the human heart has shown that
there exists a mechanism which may perhaps help to elucidate the manner
in which these hitherto imperfectly explained transferences of activity are
brought about, and although the details have not all been worked out, it may
perhaps be of use to place the facts on record.
It is well known that in a series of sections made through the auriculo-
ventricular junction an outstanding feature is the large number of nervous
structures present. It is no uncommon thing to find from 20 to 30 nerve
trunks cut across, some 50 uw to 100 w in diameter, most of them lying in the
fat and connective tissue of the groove, whilst in addition to these there are
trunks of large size lying amongst the muscular tissue, and apparently
derived directly from those in the groove.
200 Prof. A. F. S. Kent.
In close association with these nerve fibres there exist nerve cells,
occasionally single, often in groups of two or three, sometimes in large
numbers. Many of these cells are of great size, and in other particulars
remarkable in appearance. They are very markedly irregular in their
distribution, and may be scanty even where nerve fibres are abundant,
whilst in other situations they may be numerous.
The exact function of these nerve cells is open to conjecture, and it is
therefore of interest to find them associated with other structures, the
connections of which may throw light upon their mode of action.
The structures referred to are found lying in the connective tissue
between the auricular and ventricular muscle, and are of a type hitherto
undescribed in the heart. They consist of an elongated body, the first
indications of which in any series of sections is the appearance of two
or three nerve fibres lying amongst the fibrous tissue. In sections passing
directly from auricle to ventricle and taken vertically to the surface, these
fibres are as a rule cut transversely. If the series be followed the number
of fibres in the group is seen to increase, until ultimately a large number are
present.
At this point some resemblance to an ordinary nerve trunk is presented,
and the diameter of the structure may be about 170 yp.
The constituent fibres vary a good deal in size, ranging from about 3 pu to
about 12m in diameter. A number of measurements gave the average
diameter of the most numerous fibres as about 7 ~. Connective tissue of
the fibrous variety is present in the bundle, but is principally developed at
the periphery, where in the greater portion of the length of the bundle, a
definite sheath is present, of considerable thickness, and composed of many
layers with large lymphatic spaces lying between them.
If the bundle, for the structure has now assumed the form of a bundle,
be followed further, a new constituent will be noticed to have made its
appearance. This new constituent is muscle, and it generally appears as a
small mass of tissue which stains more deeply than the rest of the bundle,
and is readily distinguished from the other constituents present.
The muscle, after its appearance in the bundle, is generally to be found as
a fibre running longitudinally, or winding amongst the other tissues, and
showing a sharp differentiation into the darkly staining sarcostyles, and the
lighter sarcoplasm. The latter is generally arranged at the centre of the
fibre, whilst the sarcostyles occupy the periphery.
One muscle fibre having appeared, others are soon noticeable, and the
number increases until a considerable portion of the bundle may be occupied
by muscular tissue. .
Neuro-Muscular Structures in the Heart. 201
At the same time that the muscle becomes prominent in a bundle it may
be noticed that a very large increase in the blood supply has taken place,
and that, instead of the occasional small vessel observed at the commence-
= —
Z
\
Uf:
* BB! y
Gio:
Transverse section of structure lying in the connective tissue between the auricle and
ventricle of human heart. Xabout 300. c.7/.s., connective tissue sheath ; /.s., lymph
space ; b.v., blood vessel; v.f., nerve fibres; m., muscle. The general relations of
parts only are shown, minute detail being for the most part omitted.
ment, a rich supply of blood is brought to the bundle by vessels of
considerable size, and distributed through its substance. It may be stated
generally that the blood supply is found to be most abundant in those
202 Prof. A. F. 8S. Kent.
situations where the muscular tissue forms a constituent portion of the
bundle.
With regard to the character of the muscle fibres which are found in the
bundle, these appear to be of two kinds. Where the muscle is well
represented some fibres will usually be found to resemble the tissue present
in the neighbouring chamber of the heart. Others, however, are of a
different type, and appear as large pale fibres, with but few sarcostyles, those
that are present being grouped around the periphery of the fibre.
If the series of sections be followed further it will be found that the
bundle, which at first may be at some distance from the muscle of the heart
chamber, gradually approaches this latter, whilst at the same time some of
the large pale fibres alluded to as being present in the bundle will be
observed to be making their appearance also in the auricular or ventricular
tissue, and, after a time, a definite exchange of muscle fibres on a large scale
will be observed to take place between the auricle, or ventricle, and the
bundle. Fibres which are apparently normal auricular or ventricular tissue
approach the connective tissue, pass through as a definite mass of tissue,
penetrate the connective tissue sheath of the bundle, and come to lie in its
interior. In many cases the amount of muscle entering the bundle in this
way is considerable, and much of the tissue is indistinguishable from
ordinary cardiac tissue. Some, however, is of the character already
described, consisting of fibres with clear centres and sarcostyles scattered
around the periphery, and, though smaller, presenting some of the characters
of Purkinje fibres.
It follows from this description that the amount of muscle present in the
bundle varies considerably from place to place, and, moreover, that the
character of the muscular tissue varies also, being sometimes similar to
auricular or ventricular tissue, and having similar staining properties, and
sometimes pale fibres containing much faintly staining sarcoplasm with
comparatively few darkly staining sarcostyles.
The nerve fibres which form so important a part of the structures
described are of various sizes, from 3p to 12. They run, as a rule, a
longitudinal or somewhat winding course in the bundle, and may be demon-
strated to be connected at various points with the nervous structures lying
in the fat at the auriculo-ventricular junction. They may also be traced
leaving the bundle at various points, and passing away through the connective
tissue towards the neighbouring tissues.
From the description which has been given it is apparent that the
structure which has been described presents itself as an elongated body of
different diameters at different parts of its course, and therefore of a conical
Neuro-Muscular Structures in the Heart. 203
or fusiform shape, into the composition of which both nerve fibres and
muscle fibres of two distinct varieties enter, surrounded by a distinct con-
nective tissue sheath in which large lymphatic spaces exist, and being
abundantly supplied with blood. Further, that these structures can be
shown to be connected with the muscular tissue of the auricle or of the
ventricle on the one hand, and with the nervous structures lying in the
auriculo-ventricular groove on the other.
Tt will be at once apparent that the structures described have many
points in common with the neuro-muscular spindles found in skeletal
muscle. The association of nerve and muscle fibres in a definite structure,
the modification of the muscle fibres, the general shape, even the connective
tissue sheath with its lymphatic spaces, all recall the structure of neuro-
muscular spindles.
And it may be that, just as the structure of the two organs is similar, so
also are their functions. In the neuro-muscular spindles‘of skeletal muscle
we see organs destined for the reception of impulses from the muscle fibres—
impulses which pass to a centre consisting of nerve cells and throw it into
activity, ze. the organ is a receptive one and functions as a part of a
reflex are.
In the neuro-muscular structures described we see organs which from
their structure and connections may well function as receptive organs,
which may well be roused to activity by the muscle, and transmit impulses,
it may be, to the local centre, ie. to the nerve cells in the auriculo-
ventricular groove.
And, further, if this is so, it is difficult to avoid the suggestion that we
see here, as yet imperfectly described and obscure in some of its workings,
a local mechanism whose function it is to place in communication the various
chambers of the heart, and to correlate their activities—a mechanism con-
sisting of receptive organ, afferent path, centre, efferent path, and distributing
organ, and constituting a local reflex arc, which may perhaps exhibit only
an occasional activity, the co-ordination of the cardiac rhythm being, as a
rule, provided for by the muscular connections of the auriculo-ventricular
bundle, but which may be capable of controlling that co-ordination when the
bundle is no longer perfect.
This research has been assisted by a grant from the Research Fund of the
University Colston Society.
204
bs
Neuro-Muscular Structures in the Heart.
REFERENCES.
Braeunig, K., “ Ueber musculiése Verbindung zwischen Vorkammer und Kammer
bei verschiedenen Wirbeltierherzen,” ‘ Archiv f. Anat. und Phys.,’ 1904, Phys.
Abth., Suppl. ;
Hill, Leonard, ‘ Further Advances in Physiology,’ 1909, p. 61.
His, W., junr., “Die Thitigkeit des embryonalen Herzens und deren Bedeutung
fiir die Lehre von der Herzbewegung beim Erwachsenen,” ‘ Arbeiten aus der
medizinischen Klinik, Leipzig, 1893.
Holst and Monrad Krohn, ‘Quart. Med. Journ.,’ vol. 4, p. 498.
Kent, A, F. Stanley, “ Researches on the Structure and Function of the Mammalian
Heart,” ‘ Proc. Phys. Soc.,’ 1892, vol. 6; London, St. Mary’s Hospital, Novem-
ber 12, ‘Journal of Physiology,’ vol. 14, No. 1.
Kent, A. F. Stanley, “‘ Researches on the Structure and Function of the Mammalian
Heart,” ‘ Journal of Physiology,’ vol. 14, Parts IV and V.
Details of these investigations were also given as follows :—
“On the Mammalian Heart.” Research presented for the Rolleston Memorial
Prize, Oxford, February, 1892.
“Some New Points in the Structure of the Mammalian Heart.” Read before
the Biological Club, Oxford, March 25, 1892.
“ Wurther Researches on the Structure and Function of the Mammalian Heart.”
Communicated to the British Medical Association, Neweastle-on-Tyne, 1893.
Kent, A. F. Stanley, “On the Relation of Function to Structure in the Mammalian
Heart,” ‘St. Thomas’s Hospital Reports,’ 1893, vol. 21.
Kent, A. F. Stanley, “The Structure and Function of the Mammalian Heart,”
‘Brit. Assoc. Reports,’ 1894, p. 464.
Krumbhaar, ‘ Archiv Inter. Med.,’ June, 1910.
Lewis, Thos., ‘The Mechanism of the Heart Beat,’ London, 1911, p. 3.
Martin, C. F., and Klotz, Oscar, ‘Amer. Journ. Med. Sci.,’ 1910, vol. 140, p. 216.
Price and Ivy Mackenzie, ‘ Heart,’ 1912, vol. 3, p. 233.
Retzer, R., “ Ueber die musculése Verbindung zwischen Vorhof und Ventrikel des
Siiugethierherzens,” ‘ Archiv f. Anat. und Phys.,’ 1904, Anat. Abth.
Starling, E. H., ‘ Principles of Human Physiology,’ London, 1912, p. 1006.
Tawara, S., ‘Das Reitzleitungssystem des Siugetierherzens,’ Jena, 1906.
205
The Alleged Excretion of Creatine in Carbohydrate Starvation.
By Grorcre GrauamM, Beit Memorial Fellow, and E. P. Poutton, Radcliffe
Travelling Fellow.
(Communicated by Dr. F. G. Hopkins, F.R.S. Received August 5,—
Read November 20, 1913.) z
(From the Pathological Department, St. Bartholomew’s Hospital, and the Physiological
Department, Guy’s Hospital.)
CONTENTS.
PAGE
Ninibreductionbar. ssf een tecetaise Soeee Aeneas opisa sna’ Mt ceesunsedachseeesoasaeaes 205
I. The Effect of Aceto-acetic Acid on the Estimation of Creatinine...... 206
II, A Method for Removing Aceto-acetic Acid from Urine preliminary
LORpNesHshimationyon Crea vimin etaeseee sce saensters= eee aeeerseer eee eree 212
Ili. The Alleged Excretion of Creatine on a Carbohydrate-free Diet...... 216
Introduction.
It was stated by Cathcart (4) and Benedict and Myers (2) independently,
in 1907, that creatine was excreted in the urine during inanition. Cathcart (5)
has further stated that the output of creatine, caused by fasting for 36 hours,
is diminished as soon as a diet consisting of carbohydrates is taken, whereas
it is increased by a fat diet.
Rose and Mendel (19) confirm these results, laying great stress on the fact
that carbohydrates play a very important rdle in preventing the excretion of
creatine in the urine.
In the course of a 10 days’ experiment on one of us (G. G., 10), where the
diet was restricted to protein and fat and was of insufficient calorie value, we
found that no creatine was excreted in the urine. The explanation of this
discrepancy was not fully investigated at that time, but recent work by
Greenwald (12) has suggested a possible explanation.
Folin’s (8) method for the estimation of creatinine in urine depends on the
orange colour produced by the addition of picric acid and soda (Jaffé, 15).
This colour has been shown to be due to a reducing action of the creatinine
on the picric acid (Chapman, 6).
Among the reducing substances which also give a similar colour are
acetone, aceto-acetic acid, and 8-oxybutyric acid, all of which may be present
in urine under different conditions.
Van Hoogenhuyze and Verploegh (13) and Krause (16) stated that urine
to which acetone had been added produced, with picric acid and soda, a
VOL LXXXVII.—B Q
206 Messrs. G. Graham and E. P. Poulton. The Alleged
slightly darker colour than was obtained with the urine alone, but that
the colour soon faded and caused no error in the determination of
creatinine. Krause (16), Wolf and Osterberg (22), and Rose (20) found
that the addition of the ethyl ester of aceto-acetic acid to urine did not
produce any error in the estimation of creatinine, unless large amounts
(z.e. over 1 per cent.) were added. They seem to have assumed that the
action of aceto-acetic acid would be the same as that of the ester.
Recently, however, Greenwald (12), working on diabetic urines, has shown
that if aceto-acetic acid is added to urine directly a considerable error is
introduced into the estimation of creatinine.
This observation may possibly explain why we did not find any creatine in
our experiment on the fat diet. Folin (8) originally stated that acetone
and aceto-acetic acid gave the orange colour with picric acid and soda,
but remarked that they could easily be removed from the urine. In our
experiment we removed the aceto-acetic acid as far as possible from the
urine before making the estimation, in order to get rid of any disturbing
effect that the acetone bodies might have on the creatinine figures.
In the experiments described in this paper we have studied this question
in greater detail and also the means of overcoming the difficulty.
I. The Effect of Aceto-acetic Acid on the Estimation of Creatinine.
The different intensities of colour produced by §-oxybutyric acid, acetone,
aceto-acetic ester and aceto-acetic acid when treated with picric acid and
soda were first investigated.
The importance of aceto-acetic acid is emphasised by the experiments of
Arnold (1), Emden (7), and Hurtley (14). These investigators have,
independently, pointed out that in cases of acidosis the fresh urine contains
only small amounts of acetone, while aceto-acetic acid may be present in
large amounts.
Throughout our experiments the estimation of the creatinine was performed
in the usual way; 15 cc. of a saturated solution of picric acid and 5 cc. of
10-per-cent. caustic soda were added to 10 cc. of urine, the mixture was
allowed to stand for seven instead of five minutes, and then diluted to
500 ¢.c. with water. Folin (8) stated that the maximum intensity of colour
occurs in five to nine minutes after mixing. We have always waited seven
minutes because we found that five minutes was not always sufficient if
the urine was slightly diluted, as occurs in the estimation of the
creatinine+ creatine by this method. The matching was done with a
Duboseq colorimeter against an N/2 potassium bichromate solution. All
the matching was done by E. P. P. while the scale was read by G. G., six
Excretion of Creatine in Carbohydrate Starvation. 207
to eight readings were made and the mean was taken. The total nitrogen
was estimated by Kjeldahl’s method. The aceto-acetic acid+acetone was
estimated by the Messinger-Huppert method.
The substances tested were prepared in the following manner, and we wish
to thank Dr. Hurtley for very kindly supplying us with them. The
f-oxybutyric acid was extracted from urine and the strength of the solution
was accurately known; the solution was nearly colourless. The acetone
was chemically pure. The ethyl ester of aceto-acetic acid was obtained
by distilling the pure commercial ester under reduced pressure, and the
product boiled constantly at the correct boiling point for the pure substance.
The aceto-acetic acid was obtained from the ester, which was hydrolysed
by adding the theoretical amount of normal caustic soda, and allowing it to
stand at the room temperature for 36 hours, when the hydrolysis was
complete. It will be seen that the solution used consisted of the sodium salt
of aceto-acetic acid and an equivalent amount of ethyl alcohol. The mixture
was diluted and the amount of acetone present was determined by Folin’s
method (9). The amount of aceto-acetic acid corresponding to the acetone
present was deducted from the theoretical amount of aceto-acetic acid in
order to get the correct value for the aceto-acetic acid. The aceto-acetic
acid was kept in an ice chest in order to prevent its decomposition, and was
tested from time to time by means of the Folin and Messinger-Huppert
methods.
8-oxybutyric acid, acetone, the ethyl ester and the sodium salt of aceto-
acetic acid all give an orange colour when treated alone with picric acid and
soda, but on dilution the solution is much paler than the usual colour
obtained with urine under these conditions. These substances were then added
to urine In varying concentrations, and the colour produced by the addition
of picric acid and soda was compared with the colour produced by the urine
alone with the picric acid and soda, without the addition of these substances.
The addition of 8-oxybutyric acid produces practically no alteration in the
colour obtained by adding picric acid and soda to urine.
Thus when added to the urine (Table I) in amounts corresponding to
0036 grm. per 100 cc. and 1 grm. per 100 ce. it caused no error at all
in the creatinine determination. When present in amounts corresponding
to a 2°16-per-cent. solution it made the colour slightly lighter, causing a
difference of 0°27 mm. scale reading, which is almost within the limit of
accuracy of the method. The amounts of the @-oxybutyric acid added
are quite comparable with those found in the urine in diabetes, and as the
error caused even by large amounts is so small its effect may be safely
neglected.
ay 2
208 Messrs. G. Graham and E. P. Poulton. The Alleged
Table I shows the Effect of Increasing Amounts of 8-oxybutyric Acid,
Acetone and Aceto-acetic Ethyl Ester on the Determination of
Creatinine in Urine.
|
C : Error in the
| een | an eae Scale reading Creatinine, determination of
| COKE | cn in mm. erm. per 100 c.c. creatinine,
| grm. per 100 c.c.
|
8-oxybutyric acid added to urine.
0) 0 7 0-116
0-036 0°54 7 0-116 ) |
1 | 15°0 6-88 0°118 +0 °002 |
2°16 | 32 °4 HM O°1ll —0 005
Acetone added to urine.
0 | (0) 7 0-116
0-04 | 06 of oil 0°114 +0 002
0°17 | 2°5 7 0-116 0 |
1 15 76 0-106 —0°01
| 16 | 24, 8-24 0-098 —0-018
76 | 12 34 0-066 —vU ‘05
| Aceto-acetic ethyl ester added
to urine.
(6) | (a) 7 0-116
Ojet! 1°5 1 2 0-113 —0 ‘0038
| 0°5 | T3 75 (+108 —0 008
| 0°75 | 10-7 8°17 0-099 —0-017
| 1 15 6°9 0-117 +0001
2 30 6°3 07128 +0012
Acetone if added to the urine in amounts less than 0:2 per cent. (Table 1)
does not introduce any error at all. A 1-per-cent. solution makes the colour
lighter than usual, while if the acetone is present in larger amounts the
colour becomes much lighter and it fades very rapidly on standing. As
acetone is excreted in urine in very small amounts the creatinine determina-
tions will not be affected, as a 0°17-per-cent. solution caused no error. These
results do not agree with those of van Hoogenhuyze and Verploegh (13) and
Krause (16), who found that a 1-per-cent. acetone solution made the colour
darker, but that the error disappeared on standing.
Aceto-acetic ethyl ester when present in small quantities produces a slight
lightening of the colour. Thus a 0-1- and 0°75-per-cent. solution causes an
error in the scale reading of 0°2 and 11mm. Larger amounts, on the other
hand, cause a darkening effect, but the colour becomes much redder than
usual, which makes it really impossible to match it with the N/2 bichromate
solution. These results agree with those obtained by Krause (16), Wolf
and Osterberg (22), and Rose (20). This experiment is not of much practical
importance, as the ethyl ester is never excreted in urine, but we have made
Excretion of Creatine in Carbohydrate Starvation. 209
it because other observers have added this substance to urine instead of
aceto-acetic acid.
The sodium salt of aceto-acetic acid produces a much more marked effect
than the other acetone bodies. Even when added to urine in small amounts
the colour obtained with picric acid and soda is not darker, as stated by
Krause (16) and others, but is actually lighter, and when present in large
amounts the colour is very much lighter (Table II and fig. 1). The error
is not eliminated on standing but increases.
Table II shows the Effect of Increasing Amounts of Aceto-acetic Acid on the
Estimation of Creatinine in Urine.
|
Aceto-acetic acid added Scale Creatini Error in the creatinine
. - reatinine. oS ic
to urine. reading. determination.
| | | |
grm. per grm. per 24 hrs. grm. per | grm. per
100 c.c. in 1500 c.c. 100 c.c. 100 c.c. | per cent.
0) a) ti 0-116 | (0)
0 0234 0°35 7°33 0-111 | 0-005 | 4°3
0 0468 0-702 8 | 0-101 | 0-015 | 12-9
0-093 1°4 9 0-09 0-026 22 -2 |
0-187 2°8 11 °3 0-072 } 0-044. 38
} 0°374 5°6 | 14°66 0-055 0-061 52 6
' Thus if the concentration of the sodium aceto-acetate is only 0°0254 or
0:0468 per cent. the creatinine estimation is too low, the actual errors being
0005 and 0:015 grm. respectively. The error produced by larger amounts
is very striking, for if the concentration is increased to 0°374 per cent. the
error is as great as 0°061 grm., and the percentage error in this case is
52°6 per cent. As amounts of aceto-acetic acid up to a concentration of
0-4 per cent. may be excreted in diabetes, the error caused in such cases
must be very great. If the error in the creatinine determination be plotted
against the concentration of the aceto-acetic acid the resulting curve is
almost a straight line (fig. 1).
The chemistry of this action is at present engaging our attention.
It was not possible to isolate the pure acid and add it to urine, but this
does not matter, as the aceto-acetic acid is excreted in the urine partly as
the free acid and partly as a salt. Moreover, in the process of estimating
the creatinine an excess of caustic soda is added, and this must convert all
the free acid into the sodium salt.
The solution of sodium aceto-acetate used in our experiments also con-
tained 0°37 grm. ethyl alcohol to 1 grm. of aceto-acetie acid.
The presence of ethyl alcohol in the sodium aceto-acetate solution (due tu
its mode of preparation from aceto-acetic ethyl ester) is a possible disturbing
210 Messrs. G. Graham and E. P. Poulton. The Alleged
factor, as it might be the cause of the colour change. However, the addition
of alcohol to urine in amounts which correspond to those added with the
sodium aceto-acetate did not produce any alteration at all in the colour.
The possibility still remained that it was the mixture of the alcohol with
the sodium aceto-acetate which was responsible for the change in colour.
There is no means of directly disproving this hypothesis, as the alcohol
cannot be removed from the sodium aceto-acetate solution without destroying
the salt. There is, however, indirect proof that this is not the case, as will
be shown in the following paragraphs.
Aceto-acetic acid is excreted in the urine during carbohydrate starvation.
As will be shown later on, the aceto-acetic acid can easily be removed from
the urine without breaking down creatinine or creatine, and this procedure
was followed in three diet experiments which will be described in detail
later on. The urine which contained aceto-acetic acid had apparently less
creatinine in it than the urine from which the aceto-acetic acid had been
removed. Thus on the 2nd day Experiment I, a concentration of aceto-
acetic acid of 0-065 per cent., caused an error in the creatinine figure of
0-017 grm. per 100 c.c., and a concentration of aceto-acetic acid of 0-081 per
cent. on the third day caused an error in the creatine of 0:028 grm. per
100 c.c. On the second and third days of Experiment II the concentration
of aceto-acetic acid of 0085 and 0-112 per cent. produced errors of 0:°018 and
0:027 erm. per 100 c.c. respectively (Table ITT).
Table I1I shows the Error caused in the Estimation of Creatinine caused by
the Excretion of Aceto-acetic Acid in the Urine consequent on
Carbohydrate Starvation. (Extracted from Tables VII-IX, pp. 217
and 218, of this paper.)
Error caused by
aceto-acetic acid
in determination
of creatinine.
| Concentration of
Day. aceto-acetic acid
per 100 c.c.
grm. per 100 c.c.
Diet, Experiment I—
2
cake Silas pdau emeert rine oe 0-065 0017
SiensMnts spate eee oe Cene 0-081 0-028
Diet, Experiment II—
Sad doco! agahopagoons soodeauaK 0-029 | 0-005
Pete sapobnEpeAdoadsesesdcoNsne 0-085 0018
SS Pian SAN AER ra RE LER 0c 0-112 0-027
ee aasr conn atnnence aatiermco cee 0-036 0-01
Poa Ae Ban BOOEA CNN BRSCUDOROG 0072 0-008
Excretion of Creatine in Carbohydrate Starvation. 211
These figures have been plotted on the fig. 1 previously referred to and they
lie fairly close to the curve. The figures for the second day of Experiment I
and for both days of Experiment II lie somewhat below the curve, while the
figure for the third day of Experiment I lies a little above the curve, but the
difference is in no case great.
0:05 O14 O15 o2 025 0:3 035 04%
Fic. 1.—The curve shows the error in grammes per 100c.c. in the estimation of creatinine
caused by increasing amounts of aceto-acetic acid. Abscisse: percentage concen-
trations of aceto-acetic acid. Ordinates: the error in the creatinine determinations
expressed in grammes per 100 cc. © = the error in the creatinine determinations
on second and third days of Expt. I (Table VII). ( = the error on first, second,
and third days of Expt. II (Table VIII).
It must be remembered that the figure for the concentration of the aceto-
acetic acid in the diet experiments was obtained by the Messinger-Huppert
method, which makes no distinction between acetone and aceto-acetic acid. It
is true that acetone is only present in urine in small amounts, but in consider-
ing the effect of the aceto-acetic acid some allowance should be made for the
amount of acetone present. A diminution in the concentration of the aceto-
acetic acid would make the points below the curve more nearly approximate
to the curve but would displace the point above the curve away from the
curve.
The general agreement between the errors produced by aceto-acetic acid in
these experiments, and the errors produced when sodium aceto-acetate con-
taining ethyl alcohol is added to urine, point to the conclusion that it is the
aceto-acetic acid itself and not the alcohol that causes the errors, in the latter
case.
Our experiments agree with those of Greenwald and show that the aceto-
212 Messrs. G. Graham and E. P. Poulton. The Alleged
acetic acid produces a considerable error in the estimation of creatinine, so
that the result obtained is too low.
II. A Method for Removing Aceto-acetic Acid from Urine Preliminary to the
Estimation of Creatinine.
In Folin’s method for estimating the creatinine+ creatine, the creatine is
converted into creatinine by heating on a water-bath for three hours with
normal hydrochloric acid. This procedure removes all the aceto-acetic acid
from the urine by converting it into acetone, which is distilled away. Thus
the aceto-acetic acid could not be detected by Rothera’s (21)* nitroprusside
test after one hour’s heating. Consequently the estimation of the creatinine
+creatine will not be disturbed by the presence of any aceto-acetic acid and
will be accurate, but the result of the creatinine estimation which is carried
out in the presence of aceto-acetic acid will be too low. Consequently the
result obtained for the creatinine + creatine will be higher than that for the
creatinine and will lead to the conclusion that creatine is present in the urine
whether this is actually the case or not.
The aceéto-acetic acid must, therefore, be removed from the urine before the
determinations are made. Greenwald (12) extracted the urine with ether for
two hours and found that the aceto-acetic acid was all removed by that
process ; the ether was subsequently removed by aération for one hour. ‘This
process involved some dilution of the urine and in order to get over this
difficulty Greenwald added twice the amount of picric acid and soda. This
method takes some time to carry out and in our experience it is better if
possible to avoid all dilution of the urine, especially when the urine is dilute
to begin with, in order to get correct results.
The method which we employed (10) is very much simpler and we have
now tested it carefully and modified it slightly (11).
1 cc. of 10-per-cent. phosphoric acid is added to 10 cc. of urine in a
boiling tube 200 mm. long and 30 mm. wide. The mixture is then heated in
a water-bath of which the temperature is between 65° and 70° ©. and at
a pressure of about 210 mm. of mercury produced by means of a filter pump.
Bumping is prevented by allowing air to bubble slowly through the liquid by
means of a capillary tube dipping into it. The temperature must not rise
above 70° C. nor the pressure fall below 210 mm. or else concentration of the
urine takes place. If the above directions are followed only a few drops of liquid
are distilled over into the receiver, as the result of three-quarters of an hour’s
distillation. At the end of this time the process is stopped and the solution
* Rothera’s test has been shown by Hurtley(14) to be a test for aceto-acetic acid as
well as for acetone.
Excretion of Creatine in Carbohydrate Starvation. 213
cooled. The mixture in the boiling tube is neutralised with 1°5 cc. of
10-per-cent. soda* and then 15 c.c. of saturated picric acid and 5 cc. of the
soda are added. The mixture is allowed to stand seven minutes and then
the contents of the boiling tube are washed. into a 500 cc. flask and diluted
up to 500 c¢.c. with water. By heating for three-quarters of an hour, the aceto-
acetic acid can be completely removed even if it is present in a concentration
of 0-2 per cent.
If the concentration of aceto-acetic acid is greater than 0:2 per cent., the
distillation must be continued for a longer time, and the complete removal of
the aceto-acetic acid must be ascertained by testing a control with Rothera’s
nitroprusside test. We have continued the distillation for one and a half
hours, and find that no error in the creatine estimation occurred.
We have tested the method in the following manner. The amount of
creatinine in a normal urine was determined, and aceto-acetic acid was then
added in varying amounts to the urine, and the creatinine again estimated.
The aceto-acetic acid was then removed by the distillation method, and the
creatinine again estimated. The results obtained show that the distillation
did not break up any of the creatinine, and that the aceto-acetic acid was
completely removed (Table IV).
Table IV shows that the Error caused by Varying Concentrations of Aceto-
acetic Acid in the Urine is completely removed by the Distillation
Method.
|
Creatinine by Folin’s method. | G@rentinineratte:
BUMICUUIO HW ACCLO nap aM teeter ene RA eS | removal of the aceto-
acetic acid added to ti an
urine. Uri Urine + aceto-acetic | eeu Cae Oy Uae.
| rine alone. AAG | distillation method.
grm. per 100 c.c. grm. per 100 c.c. grm. per 100 c.c. | grm. per 100 c.c.
0°088 approx. | 0-145 0-128 0°145
| 0044 ~—C, 0-108 0-097 0-105
0°18 | 0-096 Not estimated 0-096
0°27 0-096 0-053 0-094
One of the most important questions to decide was whether creatine was
converted into creatinine in this process of distillation.
Pure crystalline creatinet was added to normal urine in varying amounts,
* The phosphoric acid must be adjusted against the 10-per-cent. caustic soda, and the
correct amount of caustic soda necessary to neutralise 1 ¢.c. phosphoric acid must be
added.
+ We wish to thank Dr. F. G. Hopkins and Mr. Mackenzie Wallis for kindly
supplying us with the pure creatine.
214 Messrs. G. Graham and E. P. Poulton. The Alleged
and the creatinine was then determined by Folin’s method and by the
distillation method. The creatine was then converted into creatinine by
heating on the water-bath for three and a half hours or longer with 5 c.c.
normal hydrochloric acid, in order to get the creatinine+ creatine figure.
These experiments show that in no case was any creatine converted into
creatinine by the distillation, even when (Experiment 5) there was actually
more creatine (0152 grm.) than creatinine (07119 grm.) in the urine
(Table V). In the first three experiments, when creatine was present in
small amounts, practically all the creatine added to the urine was converted
into creatinine by heating for three and a half hours on the water-bath. In
the fourth experiment, when 0:076 grm. of creatine was present in the urine,
only 48 per cent. of the creatine was converted into creatinine after
three and a half hours, and even after five and a half hours only 89 per
cent. could be recovered. In the fifth experiment, with a very large amount
of creatine (0°152 grm. per 100 c.c.), only 28 per cent. was converted after
heating for three and a half hours.
Table V shows that Creatine is not broken down into Creatinine by Heating
with Phosphoric Acid in the Distillation Method.
emenec Creatinine. Creatinine | Creatine
iearieeriac + recovered. Creatine Pa
iNowofs |) eoace creatine in the added to
as : : centage
expt. eae Wire calculated | estimation | urine as
creatinine Folin’s Distilla- Ba a pei ie recovered.
added to imothod tion overt tini ;
ani A method atinine. | creatinine.
|
grm. per grm. per | grm. per grm. per grm. per grm. per
100 c.c. 100 c.e. 100 e.c. 100 c.c. 100 c.c. 100 c.c
] 0:01 0:071 0:071 0 :081+ 0:01 0:01 100
2 0-016 0 095 0-096 0 -110+ 0:015 0:016 95
3 0 :034 0°112 0'113* 0°145+ 0-033 0 °034 97
0°154+ 0 :037+ 47 °5
4 0:076 0-117 0°117 \ 0-185f 0 oss 0-076 89 °5
0 °183§ 0 :063§ 83
5 0152 0°119 0:120 0°1638t 0-044, 0-152 24
* Distilled 14 hours.
+ Heated for 3} hours on the water-bath.
ae ” 53 ” ”
§ By the autoclave method.
Benedict and Myers (3) have also noticed that the water-bath method
gave an incomplete result. when urine containing 0:066 erm. creatine per
100 cc. was used. Mellanby (18) has pointed out that five hours’ heating
on the water-bath is usually necessary to get a complete conversion.
However, our observations show that three and a half hours on the water-
Excretion of Creatine in Carbohydrate Starvation. 215
bath is sufficient to estimate accurately small amounts of creatine up to
0:034 grm. per 100 c.c., but that a greater quantity than this cannot be
converted quantitatively into creatinine in three and a half hours. Larger
amounts than 0-034 grm. per 100 cc. must be heated for more than three
and a half hours, and it is very difficult, even so, to convert all the creatine
into creatinine. As the greatest amount of creatine found by Cathcart (5)
in a case of carbohydrate starvation was 0°38 germ. per day, three and a half
hours’ heating on a water-bath would be quite sufficient to convert prac-
tically all the creatine, that might be present in the urine, into creatinine.
Finally, to test the accuracy of the distillation method, we have employed
it for urines containing both creatine and aceto-acetic acid in solution
together (Table VI). In one experiment creatine was added to normal
urine, and the estimations were carried out with and without the addition of
aceto-acetic acid. In the second experiment the creatine was added to part
of the urine of the third day of the diet experiment (Table VIII). In this
case the urine already contained aceto-acetic acid. The results (Table V1)
show that, even when both aceto-acetic acid and creatine are present at the
same time, the aceto-acetic acid can be removed from the urine without
breaking up the creatine, and that the creatine, if present in small amount,
can be converted almost quantitatively into creatinine after three and a half
hours’ heating. The series of control experiments shows that the distillation
method gives satisfactory results.
Table VI shows the Accuracy of the Distillation Method for Urines
containing both Creatine and Aceto-acetic Acid.
cneeieS|
Creatinine. Cr etn Creatine
| i er ine | yecovered P t
| |- i (as lie xara ercentage
| | j Getae atinine), | ment (as recovered.
| Folin’s | Distillation) Folin’s | aa
method. | method. method. | SAEED)
| grm. per grm per grm. per grm. per
| 100 e.c. 100 ¢.c. 100 c.e. 100 c.c.
A | Normal urine + creatine 0-071 0-070 0 ‘080 0:01 100
| 0-01 grm. per 100 c.c.
| Same _—iurine + creatine 0-047 0-070 0:079 0-009 90
0:01 grm. per 100 c.c.
+0°06 grm. of aceto-
acetic acid per 100 c.c. |
B | Urine of Day 3, Experi- 0°10 0°119 0°119 0
| ment II, containing
| 0112 grm. of aceto-
| acetic mid per 109 c.c.
| Same urine + creatine 0°10 0-120 0 +156 0-036 90
| 0°04 grm. per 100 c.c.
216 Messrs. G. Graham and E. P. Poulton. The Alleged
In the examination of any urine which contains aceto-acetic acid, and
which is thought to contain creatine, two estimations are required, namely,
that of the total creatinine + creatine by the original Folin method, and that
of the creatinine alone by the method given in this paper.
Ill. The Alleged Excretion of Creatine on a Carbohydrate-free Dict.
It is well known that the consumption of a diet containing no carbo-
hydrates produces acidosis, with the excretion of B-oxybutyric acid, aceto-acetie
acid, and acetone. The aceto-acetic acid will cause an error in estimating
creatinine and creatine, and must be removed to get accurate results.
We have performed three diet experiments on three separate individuals,
and have investigated the creatinine and creatine excretion, taking this
precaution. The experiments were begun about 12 hours after the last
ordinary meal in Experiments I and III, and six hours after, in Experiment II.
Tables VII, VIII, and IX show the various determinations made. The
creatinine was first of all estimated directly by Folin’s method without
removing the aceto-acetic acid, and the results are referred to as “apparent
creatinine.” The true creatinine was then obtained after removing the
aceto-acetic acid by the distillation method. The creatinine+creatine was
determined by heating the urine for three and a half hours on the water-
bath with hydrochloric acid. By subtracting the apparent creatinine values
from the creatinine+creatine output, the apparent creatine was obtained,
and, by subtracting the true creatinine from the creatinine+creatine, the
true creatine output was obtained. Duplicate determinations were performed
in each case, and in each determination the mean of six to eight readings of
the scale was taken.
In Experiment I (E. P. P.), cream alone was eaten on the first two days;
on the third day protein was added to the diet. The calorie value of the
diet was low. The effect of the withdrawal of carbohydrates was shown by
the prompt appearance of aceto-acetic acid in the urine. On the first day
the nitroprusside reaction (Rothera’s) was faint, but on the second and third
days it was well marked, and 0:872 and 0°874 erm. of aceto-acetic acid were
excreted. On the first day the apparent creatinine was 1:82 grm., while
the true creatinine and creatinine+creatine was 1°78 and 1°80 respectively,
so that no creatine was excreted in the urine, as the difference is within the
limits of experimental error. On the 2nd day the apparent creatinine was
diminished to 1°58 grm., while the true creatinine and creatinine + creatine
were practically the same as on the previous day, 2.¢., 1°81 and 182 grm. The
apparent creatine was, therefore, 0:24 erm., while no true creatine was excreted.
On the third day the apparent creatinine had fallen to 1-42 grm., while the
true creatinine and creatinine+creatine was still 1°72 germ.
Excretion of Creatine in Carbohydrate Starvation.
217
The apparent
creatine had, therefore, increased to 0°31 grm., while as a matter of fact
no true creatine was excreted.
In Experiment II (G. G.) ? pint of cream and two eggs were eaten on each
day. The amount of aceto-acetic acid excreted was greater than in Experi-
ment I, and the nitroprusside reaction was quite strong on the first day,
0-3 grm. being excreted. On the second and third days the aceto-acetic
acid amounted to 1°06 and 1:46 grm. The true creatinine output was
slightly lower than in the case of E. P. P., but it remained equally constant
Table VII.—Experinment I.
Subject, E. P. P. Date, June 18-20, 1913.
|
After removal of
|
By the Folin method. aceto-acetic acid. | Aceto-acetic acid. |
5 tsb arch |
| Total | F True | True |
ey: Ty nitrogen. @ecatind Apparent creatine. | ~yeatinine.| creatine.
| | Apparent | “Te?vnine | | Gm. per Concentra-
| creatinine. iy 7) da | tion in
creatine. | | y- 100 e.c
| Grm. per| Grm. per Grm. per | Grm. per | ie
day. 100 c.c. day. day. |
| |
| (cca) ewerm: pene hn) erm |
1 | 740 12 T8280 0 0 Meson | 10) — | =
Zin} = OTO 13 °5 5S ee} e082 0°24 O-O18S5)) SekesT 0 0°872 | 0:065*
3 1070 16 °25 RAZ i 0°30 0-028 | Tin 0) 0 °874 0-081 |
Diet eaten.—Day 1 and 2: Cream, 300 c.c. Calorie value (approximate), 1060. }
Day 3: Cream, 300 c.c.; plasmon, 50 grm.; eggs, 2. Calorie value (approximate), 1640.
* On this day the volume of urine was small and an equal volume of water was added to it before the creatinine
determinations were made in order to get a reading on the colorimeter scale within the limits advised by Folin.
This
dilution will halve the concentration of the aceto-acetic acid in the solution used for the Folin estimation, which
becomes actually less than that of the succeeding day.
Table VI1I.—Experiment II.
Subject, G.G. Date, June 22-25, 1913.
Day. |
wOnNnre
Volume.
By the Folin method.
After removal
of aceto-acetic acid.
|
Aceto-acetic acid.
Total
| nitrogen.
Diet eaten.—Cream, 400 c.c.; eggs, 2.
|
_ Apparent creatine. | |
Creatinine | vill Concen-
Boats iret | Caan | ieee | ae pet | tration per
re ue: | creatine. | Grm. per| Grm. per yl Sal y: 100 c.e.
| | day. 100 c.c.
|
|
grm. grm. grm. | grm.
1-46 153 0:07 0 0053 1°52 0 0°3 0 029
1°21 143 | 0°22 0:0175 1°43 10) 1-06 0 :085
aes 1°53 0°36 0°0274 | 1°52 | 0 1°46 0°112
Calorie value (approximate), 1400.
218 Messrs. G. Graham and E. P. Poulton. The Alleged
Table [X.—Experiment III. Subject, M.D. Date, June 25-28 1913.
|
By the Folin method. After removal of Aceto-acetic acid.
| aceto-acetic acid.
Day. | Volume. Settee | +e Apparent creatine.
| Apparent ee | ‘True True Grm, Naan
creatinine. | | oatine. Grm. | Grm. per| creatinine. | creatine. | per day. Aen .
per day. | 100 c.c.
c.c. grm. erm. grm. | |
1 600 11-99 1°93 2°05 0°12 0-010 2-03 0 0°43 0 -036*
2 750 14°43 2-02 2°14 0°12 0-008 PAS a | 0 0-99 0 -066*
3 1220 14°82 2:09 2°25 0°16 —_ 2°27 0 = =
Diet eaten.—Cream, 500 c.c. ; eggs, 3. Calorie value (approximate), 1600 calories.
* On these days the volume of urine was small and an equal volume of water was added to it before the creatinine
determinations were made in order to get a reading on the colorimeter scale within the limits advised by Folin. This
dilution will halve the concentration of the aceto-acetic acid in the urine.
throughout the experiment, viz.,1‘5 grm. On the first day the apparent
creatine was already 0:07 grm., and on the second and third days it had risen
to 0°22 and 0°36 grm. respectively, but no true creatine was excreted at all.
The difference in the scale reading between the apparent and true creatinine
was 2 mm.on the third day of this experiment. It has been previously
shown (p. 211, fig. 1) that the error in the estimation of the creatinine caused
by the aceto-acetic acid in these two experiments agrees fairly closely with
the error caused by adding the same concentration of a sodium aceto-acetate
solution to normal urine.
As it was necessary to be absolutely certain that if any creatine was
present in the urine it would be converted into creatinine by the methods
we have used, some pure creatine was added to a part of the urine of the
third day in Experiment IJ. The estimation of the creatinine + creatine in
the plain urine and in the urine to which creatine had been added was
carried out under precisely similar conditions on the same water-bath.
The result (Table VI) showed that in the plain urine no creatine was
converted into creatinine, but that the creatine was almost quantitatively
converted into creatinine in the sample of urime to which creatine had been
added. This control experiment shows that creatine if present in the urine
is detected ard estimated by the methods employed.
As we wished to confirm the results of these experiments on ourselves,
Dr. M. Donaldson very kindly took the following diet for three days, viz.,
? pint of cream and three eggs each day. We wish to express our thanks
to him. The urine gave Rothera’s nitroprusside test on the first day, and
this reaction was well marked on the second and third days. The true
Excretion of Creatine in Carbohydrate Starvation. 219
creatinine in the urine was again very constant for the three days, lying
between 2 and 2°3 grm. On the first and second days the apparent creatine
was 0°12 erm., and on the third day it was 0:16, while no true creatine was
excreted. The apparent creatinine was not so low as in Experiments I and II,
but its difference from the true creatinine was quite definite enough to be
measured on the colorimeter.
Discussion of Results.
These three experiments show that the removal of carbohydrate from the
diet causes an excretion of aceto-acetic acid in sufficient amount to cause an
error in the estimation of creatinine, so that the results are too low and
creatine is apparently excreted.
Cathcart (5), Benedict (2), Mendel and Rose (19) state that creatine occurs
in the urine under somewhat similar conditions to those under which we have
worked. The amounts of creatine they obtained were about the same as
those apparently obtained by us, before we removed the aceto-acetic acid, e.g.
the largest amount that Cathcart (5) found on a fat diet was 0°38 erm., which
is slightly more than the apparent creatine we found on the third day of
Experiment: IT.
Cathcart remarks that the creatinine excretion diminishes to a certain
extent, as the result of a fat diet. We have found that it remains constant
throughout, and in the case of G. G. agreed very closely with the amount of
creatinine excreted 15 months before on a pure fat and carbohydrate diet (10).
- However, the error caused by the presence of aceto-acetic acid in the urine
results in less creatinine being found than is actually present.
Our experiments extended over about the same time as those of Cathcart,
but there was a slight difference, viz., that we had no preliminary starvation
day. However in Experiment I the condition of semi-starvation was really
very similar to that of one day’s complete starvation, as only half a pint of
cream was taken and the calorie value was 1060.
We have also published a case of carbohydrate starvation (10) lasting for
10 days in which the diet had a calorie value of only 1969 per diem. No
creatine was excreted at any time, and the creatinine excretion remained
constant throughout.
From these results we draw the conclusion that mere carbohydrate starva-
tion itself does not cause an excretion of creatine in the urine.
Naturally, no conclusion can be drawn from these experiments as to whether
creatine is excreted during prolonged periods of total starvation, but we
maintain that in all those many physiological and pathological conditions in
which acetone bodies are excreted in the urine, the estimations of creatinine
220 Alleged Kxcretion of Creatine in Carbohydrate Starvation.
and creatine must be inaccurate, unless the precaution is taken of removing
the aceto-acetic acid from the urine.
Conclusions.
1. The presence of aceto-acetic acid always causes an error in the estimation
of creatinine and the error increases with increasing amounts of aceto-acetic
acid. As the result of this error the estimation of creatinine will be too low.
This error is not eliminated if the diluted urine is allowed to stand for
varying lengths of time before making the readings.
2. The aceto-acetic acid is removed in the estimation of creatinine+
creatine and does not cause any error.
3. As the creatinine figure is too low and the creatinine + creatine figure is
correct, it will appear that creatine has been excreted.
4, Acetone and §-oxybutyric acid, if present in amounts comparable to
those which usually occur in urine, produce practically no error in the
estimation of creatinine.
5. A simple and reliable method has been devised for removing aceto-
acetic acid, preliminary to the estimation of creatinine.
6. In our experiments a carbohydrate-free diet did not cause the excretion
of any creatine.
REFERENCES.
Arnold, ‘ Zentralbl. f. Inn. Med.,’ 1900, vol. 21, p. 417.
Benedict and Myers, ‘ Amer. Journ. Physiol.,’ Boston, 1907, vol. 18, p. 362.
Benedict and Myers, ‘ Amer. Journ. Physiol.,’ Boston, 1907, vol. 18, p. 397.
Cathcart, ‘Journ. Physiol.,’ Camb., 1907, vol. 35, p. 500.
Catheart, ‘Journ. Physiol.,’ Camb., 1909, vol. 39, p. 311.
Chapman, ‘The Analyst,’ 1909, vol. 34, p. 475.
Emden, ‘Zentralbl. f. Stoffwechsel u. Verdauungs-Krankheiten, N.F.,’ 1907,
vol. 2, pp. 250 and 289.
8. Folin, ‘ Zeitschr. f. Physiol. Chemie,’ 1904, vol. 41, p. 223.
9. Folin, ‘Journ. Biol. Chem.,’ Baltimore, 1907, vol. 3, p. 177.
10. Graham and Poulton, ‘Quart. Journ. Med.,’ Oxford, 1912, vol. 6, p. 82.
11. Graham and Poulton, ‘ Proc. Physiol. Soc.,’ ‘Journ. Physiol.,’ Camb., 1913, vol. 46,
p. xliv.
12. Greenwald, ‘Journ. Biol. Chemistry,’ Baltimore, 1913, vol. 14, p. 87.
13. Wan Hoogenhuyze and Verploegh, ‘ Zeitschr. f. Physiol. Chemie,’ 1908, vol. 57,
p. 161.
14. Hurtley, ‘ Lancet,’ London, 1913, vol. 184, p. 1160.
15. Jaffé, ‘ Zeitschr. f. Physiol. Chemie,’ 1886, vol. 10, p. 399.
16. Krause, ‘Quart. Journ. Exper. Physiol.,’ London, 1910, vol. 3, p. 289.
17. Mellanby, ‘Lancet,’ London, 1911, vol. 2, p. 8.
18. Mellanby, ‘Journ. Physiol.,’ 1907-1908, vol. 36, p. 447.
19. Mendel and Rose, ‘Journ. Biol. Chemistry,’ Baltimore, 1911-1912, vol. 10, p. 213.
20. Rose, ‘Journ. Biol. Chemistry,’ Baltimore, 1912, vol. 12, p. 73.
21. Rothera, ‘Journ. Physiol.,’ Camb., 1908, vol. 37, p. 491.
22. Wolf and Osterberg, ‘Amer. Journ. Physiol.,’ Boston, 1911, vol. 28, p. 71.
sw Gd Cu ES CO oS
221
On Medullosa pusilla.
By D. H. Scort, LL.D., D.Sc., For.Sec. B.S.
(Received August 12,—Read November 20, 1913.)
[Puate 13.]
In the second edition of my ‘Studies in Fossil Botany, I referred in the
following words to the fossil plant which forms the subject of this notice.
“A very small Medullosa (named provisionally Medullosa pusilla), the stem
with the leaf-bases not exceeding 2 cm. in diameter, has since been found by
Mr. P. Whalley, of Colne, Lancashire. The stem has three steles, and
agrees very closely with J. anglica, except in size.”*
In order to clear the ground for other observers, it now seems desirable
to give some further account of this fossil, with the necessary illustrations.
Though the plant differs in no important respect from the now well-known
species W/. anglica, it is of some interest, as probably the smallest Medullosa
on record.
The fossil, as Mr. Whalley informs me, comes from the Soap-stone, imme-
diately above the Halifax Hard Bed of the Lower Coal Measures. Its
horizon may thus be compared with that of the roof-nodule specimens in
other localities.
I have only two sections of the stem, sent me by Mr. Whaliey on
January 24, 1906. There are also a couple of sections received this year
which appear to be of the same plant, and perhaps of the same specimen, but
only show a leaf-base or petiole.
Both the sections of the stem are transverse, but in one of them a stele is
shown partly in longitudinal section, owing, no doubt, to displacement
(Plate 13, fig. 2, above). This has enabled me to compare the minute
structure of the wood with that of IZ anglica.
General Structure.
The extreme dimensions of the specimen as shown in the sections are
22x13 mm.; the form is much distorted, and in the direction of the
longest diameter tissue has manifestly been lost. Three leaf-bases are
present, one of which is well preserved and practically complete, while the
other two are crushed and imperfect (figs. 1 and 2). They all contain
. humerous vascular bundles, and are bounded by a “Sparganum” cortex,
* P. 441, footnote.
VOL. LXXXVII.—B. R
DIY Dr. D. H. Scott.
The best leaf-base measures about 13 mm. in the tangential and 8 mm. in
the radial direction.
The tri-stelar vascular system of the stem (fig. 3) is enclosed in a definite
but irregular ring of dark tissue, which, judging from the best-preserved
portion, is evidently an internal periderm (cf. text-fig. B). The approximate
dimensions of the region enclosed by the periderm are 7x55 mm. The
general structure is clearly the same as that of JZ. anglica, in which three
leaf-bases appear in the transverse section, and the vascular system is also
normally tri-stelar (Scott, 99, text-fig., p. 126, Plate 5, Phot. 1). In the
specimen of MW. anglica referred to, the dimensions in its present condition are
10°5x3°7 cm. The natural diameter would no doubt have been a little over
7 cm., and the other specimens investigated do not differ greatly in size. In
M. pusilla the natural diameter cannot be directly measured, as two of the
leaf-bases are crushed and incomplete. Judging from the radius of the best-
preserved portion, the true diameter must have been just about 2 cm. This gives
a proportion between JZ anglica and M. pusilla of rather more than 3°5:1.
If we compare the stelar systems, the difference is somewhat greater—that of
M. anglica in the best-preserved specimen measuring about 4x2 cm. as
against 7x 5°5 mm. in JZ pusilla, taking the periderm as the boundary in
both cases. From the means, 3 cm. and 0°625 cm. respectively, we get a
proportion of 48:1. Roughly, we may say that the linear dimensions of
M. pusilla were about one quarter of those of a typical specimen of J. anglica.
Stelar System.
The three steles are nearly equal in size, attaining a diameter of about
3mm. Little is preserved except the wood, though here and there remains
of the cambium and phloem can be found. The primary wood has a
somewhat triangular transverse section (fig. 3). It is composed for the most
part of tracheides, with comparatively little xylem-parenchyma among them.
The smallest elements, presumably protoxylem, are found at the prominent
angles, very near the outside of the primary xylem, but whether the
structure was slightly mesarch or actually exarch could not be determined
with certainty; there is evidence pointing in both directions. Similar
difficulties were met with in the case of WZ. anglica, but there, with the help
of the longitudinal sections, it was possible to obtain definite proof of
mesarch structure (Scott, 99, Plate 10, fig. 5). In JZ pusilla the partially
longitudinal section of one stele does not clearly show the position of any -
protoxylem group.
The secondary wood is very unequally developed, attaining its greatest
thickness, about 12 elements, on the inner side of the steles (fig. 3), as has
On Medullosa pusilla. 223
also been observed in M. anglica (Scott, 99, p. 89, Plate 5, Phot. 1, Plate 6,
Phot. 5). On the outer side it thins out, or may even be interrupted,
perhaps in connection with the departure of a leaf-trace bundle. The
medullary rays are numerous, with the tracheide-bands between them only
1-3 elements in width.
The longitudinal section shows something of the primary as well as the
secondary wood. In both, the tracheides have multiseriate bordered pits,
sometimes ranged in aS many as six rows. One or two narrow scalariform
or spiral elements can also be recognised in the outer part of the primary
Text-ric. A.—Approximately radial section of part of secondary wood, showing
tracheides with multiseriate bordered pits and muriform medullary rays. Drawn
by Mr. G. T. Gwilliam. x about 80. Scott Coll. 2818.
xylem. The secondary wood is cut approximately in the radial direction, and
several medullary rays are shown ; they are muriform, with very low cells;
the pits adjacent to the ray-cells are elongated radially (text-fig. A). The
wood is in all respects similar to that of M. anglica. Only one leaf-trace
bundle is shown in connection with the stelar system, and in an undivided
condition (fig. 3, /..). It measures about 650x570 yw. There is no obvious
secondary wood, and the smallest elements appear to be directed outwards,
but the preservation is imperfect.
R 2
22.4 . Dr. D. H. Scott.
_ Except for the possible absence of secondary wood from the undivided
leaf-trace—a doubtful point on which no stress can be Jaid—there is no
difference between the stelar system of JZ. pusilla and that of M. anglica.
The Cortex and Leaf-bases.
In MZ. pusilla, as in M, anglica, no sharp limit can be drawn between cortex
and leaf-base except at a level where the latter is already marked off by an
internal barrier of sclerotic strands. The appearance of such a barrier is, of
course, a preparation for the departure of the leaf-base from the stem.
In the transverse sections the best preserved leaf-base is only partly
delimited in this way; a sclerotic band runs inwards from each side, but
does not extend right across. Of the two imperfect leaf-bases, one appears
to be completely marked off by an internal sclerotic band, while the other is
not yet delimited at all (Plate 13, figs. 1 and 2)
A convenient boundary between cortex and stelar system is provided by
the periderm (fig. 3). The cortex, which contains numerous gum-canals, is
not very well preserved, but it can be seen that the vascular bundles in this
region are, on the whole, larger and rounder in section than those which have
definitively entered the leaf-base. ;
At one place a group of four or five bundles is shown, which has, to all
appearance, arisen from the division of a single primary leaf-trace (text-
fig. B). This group hes in the cortex, which is here well defined by the
sclerotic band on the exterior and the periderm within. Similar groups of
bundles formed by division are well known in the cortex of JL anglica
(Scott, 99, Plate 6, Phot. 9; Plate 11, fig. 12). In both species later stages
in the division of the bundles are found in the leaf-base itself.
The well-preserved leaf-base is best shown in the section represented in
Plate 13, fig. 2, and is here sufficiently perfect for the bundles to be counted
with approximate accuracy. There are 15 peripheral bundles (7c. on the free
side), nine on the side attached to the stem, and eight, of which two are
double, in an intermediate position. Thus the interior of the leaf-base is
poor in bundles, a condition which also exists in that of JZ anglica at a
corresponding level (Scott, 99, p. 100).
The bundles near the periphery have assumed their definitive petiolar
character, while the inner bundles more resemble those of the cortex, and
are still undergoing division. Some of the former are very well preserved
(Plate 13, tig. 4), with the phloem practically perfect; the usual exarch,
collateral structure is obvious. The sectional form of these bundles is often
slender, ze. elongated radially, as also occurs sometimes in JZ. anglica,
On Medullosa, pusilla. 225
Thick-walled elements enclose the xylem of the bundle in an interrupted,
hippocrepiform sheath, but do not extend round the phloem.
The parenchyma contains some gum-canals and presents no peculiarities.
The only differences between the leaf-base of M. pusilla and that of
M. anglica are in the hypoderma. This zone is ratber narrow in M. pusilla,
its usual thickness being about 400; in M. anglica it ranges from 2 to
Text-ric. B.—Group of vascular bundles from the cortex, probably resulting from the
division of a single leaf-trace ; pd. periderm, marking inner limit of cortex ; sc.,
internal sclerotic band, marking inner limit of leaf-base. Drawn by Mr. G. T.
Gwilliam. x about 60. Scott Coll. 2818.
3 Inm., 7.¢. if is about six times as thick, on the average, while the general
dimensions are only about four times as great. Further, in /. pusilla the
hypoderma is much simpler ; as a rule, the sclerotic strands are in a single
rank, and they never stand more than two deep (Plate 13, fig. 4). In the
leaf-base of MZ. anglica the strands are three to four deep (Scott, 99, Plate 5,
Phot. 3; Plate 12, fig. 14). In M. pusilla the principal strands are about
twice as deep as they are wide, and slightly wedge-shaped, widening
226 Dr. D. H. Scott.
outwards. In M. anglica, where they are so much more numerous, they are
quite irregular in form.
The distinction is not absolute, for in parts of the rachis attributed to
M. anglica one may find much the same arrangement as in M. pusilla.
Comparing leaf-base with leaf-base, however, there appears to be a real
difference between the two plants. The hypoderma of M. pusilla is of the
type of Renault’s Myelopteris Landrioti, var. « (Renault, 75, Plate 5, fig. 41),
while that of 1. anglica is more like his var. 8 (Joc. cit., Plate 4, fig. 29). Of
course, the agreement is far from exact, for Renault’s petioles no doubt
belonged to quite different species of Wedullosa from ours.
In the leaf-base of M. pusilla there are very few gum-canals visible in the
hypoderma, and they are not very numerous altogether (fig. 4). In JL anglica
they are very common in the hypoderma, and fairly so elsewhere. This
distinction, however, is of very doubtful value, for in a section of a detached
petiole or leaf-base probably referable to MZ. pusilla, gum-canals are numerous
in the parenchyma outside the sclerotic strands, most of the strands having
canals corresponding to them. This tissue is hardly preserved at all in the
type-specimen, so the small number of hypodermal canals observed may be
deceptive. Their distribution appears to have been a little different from
that in I. anglica, but in that species also the external parenchyma is seldom
well preserved, so comparison is difficult.
Summary and Conclusions.
If we draw up a diagnosis of I. pusilla on the lines of that given for JL. anglica
(Scott, 99, p. 111), we find few distinctions between them, apart from size.
Medullosa pusilla.—(Scott, ‘Studies in Fossil Botany,’ second edition, p. 441,
1909.)
Stem clothed by the relatively large decurrent leaf-bases.
Vascular system of stem consisting of three uniform steles. Star-rings
absent. Interior of each stele wholly occupied by primary wood.
Secondary wood of moderate thickness, most developed on the inner sides
of the steles. Tracheides (apart from the protoxylem) with multiseriate
bordered pits.
Leaf-traces probably concentric on leaving the steles, but with little or no
secondary wood, branching and becoming collateral as they pass into the
leaf-bases.
Leaf-bases, with a narrow hypoderma, consisting of a single, or locally double
series of strands, and resembling that of Myelopteris Landriotii, var. « Renault.
Gum-canals numerous in the cortex, scattered in the leaf-bases. Stem
small, about 2 cm. in diameter, including the leaf-bases.
On Medullosa pusilla. Papa
Locality: Lark Hill Pit, Colne, Lanes. Horizon: Soap-stone, overlying
Halifax Hard Bed, Lower Coal Measures.
Found by Mr. P. Whalley, 1906.
I have italicised the characters in which this form differs from JZ. anglica.
The distinctions are of little importance, with the exception of the small
size of the plant and the simpler structure of the hypoderma, points which
appear to be of some diagnostic value.
The question arises whether it is worth while to separate the species from
M. anglica on these somewhat slender grounds. The difference in size is
considerable, and not due merely to age, for secondary growth is already
fairly advanced, while the whole primary structure is on a small scale.
The specimen might, however, be from the basal part of the stem, where it
had not reached its full dimensions, or might belong merely to a dwarfed
plant. At the same time, it is perhaps equally probable that our specimen
represents a distinct species. There is strong reason to believe that the
foliage of I. anglica was of the Alethopteris type (Scott, 99, p. 102), and it is
probable that the species may have been identical with 4. lonchitica, so
abundant in the Lower Coal Measures. We have no evidence as to the
foliage of JL pusilla, but there is a certain presumption that it was also that
of an Alethopteris, both from analogy with J. anglica and from the older
observations of Renault. That author, after a careful comparison, came, as is
well known, to the conclusion that it was extremely probable that the
petioles of his Myelopteris (Myeloxylon) Landrioti bore the fronds of certain
species of Alethopteris (Renault, ’83, p. 165). In his ‘ Cours de Botanique
Fossile’ he reproduces the figure of If Landrioti, var. «, under the title
“Section Transversale d’un Pétiole d’Alethopteris” (83, Plate 28, fig. 1).
This is the section which most closely resembles the leaf-base of I pusilla.
lf IZ. pusilla was the stem of an ) —>
30
20
10
10 20 Ke) 40 50 60°C.
temperature ———>
Fig. 2.
FO) 26° 30 40 s0 60 70 &0 90 mg.
Quantity of enzyme in milltgrams.—>
Fig. 1.
enzyme necessary to produce the percentage of hydrolysis decided upon—
in this case 0°9 mgrm. for a 50 per cent. hydrolysis as shown by fig. 1—
dissolved in 10 cm.? of redistilled water. After half to one hour of contact
at the ordinary temperature the solution was introduced in portions of
1 cm.* into each of a series of eight or nine test-tubes already containing
286 mgrm. of salicin and 4 cm.? of water. The tubes were then plunged
into water-baths kept at known temperatures, and after 15 hours the
action was stopped and the proportion of glucoside hydrolysed determined as
before. The numbers obtained are set forth in Table II.
By plotting the percentage of salicin hydrolysed against the mean
temperature of the experiment these numbers give the curve indicated above
in fig. 2. The optimum temperature under the foregoing conditions is thus
found to be + 34°.
248 Mr. A. Compton. Optimum Temperature of
Table II.
Temperatures at the beginning and
end of each experiment. Salicin hydrolysed.
- per cent.
17 ‘8-17 ‘6 24 °2
29 °3-29 5 42°8
34°7 500
41 °8-41 7 43-0
50 *2-50 3 31°4
| 57 5-57 “6 16 °4
Next the activity of the enzyme was determined in the vicinity of +34°
for a 15 hours’ action with each of the following concentrations of the
substrate: M/5, M/10, M/15, M/30, and M/50, the effect of which on the
optimum temperature of the enzyme it was ultimately intended to study.
The temperature actually employed was 33°6°-33°8°; and the experimental
details were the same, except the dilutions, as already described for the
preliminary determination. The numbers obtained are given in Table III.
Table III.
Salicin hydrolysed per cent. for the following concentrations :—
Quantity of enzyme.
M/5. M/10. M/15. M/30. M/50.
mgrm.
0°5 = 31°4 35°8 28 2 —
1°0 55-1 57 °5 58 °5 49 °5 36 ‘9
2°0 79 °4 84-7 88-7 77°0 =
3°0 88 °3 93-9 97 °5 90-0 77:2
5-0 94: *2 97-5 99 °6 99 0 93 “9
7-0 94°9 96 “7 99 *2 100 °6 100 ‘0
10-0 95-7 100 °2 99-6 — 100 °5
12°5 95 °7 = 100 °6 1006 100 ‘0
These numbers give, on plotting the percentage of salicin hydrolysed
against the quantity of enzyme in play, the activity curves shown in fig. 3.
The influence of the substrate concentration on the optimum temperature
of the ferment, the fourth stage of the inquiry, may now be considered.
This is the case of determining the optimum temperature in a series of
experiments in which the concentration of the enzyme is kept constant while
that of the substrate varies. The concentration of the enzyme chosen, in
accordance with fig. 3, was 0°-4 mgrm. in 5 em.%, 1.e. 8 x 107° grm. per cm.? of
the reaction mixture. Five different solutions of the enzyme were prepared
Salicin Hydrolysis by Enzyme Action.
Salicin hydrolysed (%o) ——>
0 20 30 40
50.60 70 80 90 100
Quantity of enzyme in milligrams ———>
Fig. 3.
100
3
S
iS
20
Salicin hydrolysed (%)——>
i0 20 30 40 sO 60°C
temperature ——>
Substrate conc? M/5 to M/50
En3yme conc? 8x 10x per cm.
Fig.4-\
249
250 Mr. A. Compton. Optimum Temperature of
containing 4, 8, 12, 24, and 40 merm. dissolved in 10 cm? of water, which,
after standing for half to one hour, were introduced in portions of 1 em.’
into five series of test-tubes containing 286 mgrm. of salicin and 4, 9, 14, 29,
and 49 cm.’ respectively of water. After 15 hours’ incubation in baths
at known temperatures the action was stopped and the quantity of salicin
hydrolysed in each tube estimated as before. The numbers obtained are
given in Table IV.
Table IV.
Salicin hydrolysed per cent. with the following substrate
Temperatures at the concentrations :—
beginning and end of
each experiment.
M/5. M/10. M/15. M/30. M/50.
10°8
I
bo
im
1
o
Hd wd oan S ~7
66
10°8
bo
(or)
iin
bo A
on
bs =)
26 °5 19 -2 38 6 5
30 °0 22°3 41-9 5
== 38 °3
23 6
22 °4
19 °7 35 ‘0 43-7
15 +1
11-2
lee wast = —_— 25 °8
or
(=)
rik
oO
f=)
SdHSNHG BOIS
These numbers give graphically the curves shown in fig. 4 (p. 249).
On examination the above curves indicate, although with very different
degrees of precision, maxima in the same region of temperature. In so far
as the curves are comparable with one another, they produce the general
impression that the optimum temperature of the enzyme is constant, and
consequently independent of the concentration of the substrate. But to
answer the question more definitely curves of a uniform type, easily com-
Salicin Hydrolysis by Enzyme Action. - 251
parable among each other, are required. This can only be achieved by
_varying the concentration of the enzyme at the same time as that of the
substrate, for, ceteris paribus, the extent of an enzyme action is, as shown by
the activity curves of figs. 1 and 3, determined by the proportion of enzyme
_to substrate present in the reaction mixture. The case which constitutes the
fifth stage of the inquiry will now be considered.
Table VY.
Salicin hydrolysed per cent. with the following molecular con-
ti f . of t Se
Densty eset te centrations of the substrate and grm. of the enzyme per cm
beginning and end
of each experiment.
M/5. M/10. M/15. | M/30. M/50.
31 x 10. | 185 x 10%.) 8'7 x 10%. | 5-7 x 10™. | 5 x 1053:
— = = = 41-9
ba
Qo
it;
a
. Qo
BME NATor Bro cow
= — 50°3 | — | Sekar
6
= 70 °0
ww
co
y
(0)
iy wo
“10 DOH cr
a 68 -6 69 *4 |
a
eR
E
oo6
or)
= = 53 °0
|
or
or)
oy)
hs
oS i or
37 °7 _ — 36 °4
>
Ne)
Ve)
or
22°53 22 °*4
t 70 wr
ak uv
SS &
“”
v 60 Deo
2 2
s 1
5 50 20
>
=
<
S40 $40
s = a
] ce) x
“” 10 30 y
i $ 60
10 20. + 30 40 SO7G lO ZONDO: 40. sOo-c >
temperature—> i temperature —> Se
ri eau bstale conc? M/s. Tia6 eubstrale conc? M/I0. =
F°NEngyme conc? 31x10. gr percm> *“F-°- Enzyme conc” 13-5*10 gr percm’ iS
S
%
y
Sess
Salicin hydrolysed (4) —>
>
So
252 Mr. A. Compton. Optimum Temperature of
That the curves might be vertical enough to give sharply defined
maximum points, it was decided to aim at obtaining about 70 per cent.
hydrolysis of the substrate at the optimum temperature. A cursory
examination of fig. 3, which was constructed at approximately + 34°, shows
that to obtain such curves—assuming for the moment, what fig. 4 already
indicates the probability of, that the optimum temperature is independent
of the concentration of the substrate, and situated at about + 34°—the
quantities of enzyme required, in actions of 15 hours’ duration, are 1°55,
1:35, 1:30, 1:70 and 2°50 mgrm. respectively for the concentrations M/5,
M/10, M/15, M/30 and M/50 of the substrate. Working with these
quantities the experimental data obtained are set forth in Table V.
By plotting as before the percentage of salicin hydrolysed against the
temperature of the experiment the foregoing numbers give the curves repre-
sented in figs. 5, 6, 7, 8 and 9.
On careful examination the curves below all show that the activity of the
enzyme is greatest between +33:5° and +34:5°; in other words, that the
optimum temperature is about +34°, and is constant, notwithstanding the
wide variations in the dilution of the substrate and the accompanying
variations in the dilution of the enzyme.
_
is}
(0 20 30 40—=«S OYE
temperature ——>
Substrate conc: M/S5O.
3
8
Fig.9-|
Sakcin hydrolysed(%)—=>
10 20 SORTA OES OF lORNEZONNNN2 OSEEEsO SORC
ie temperature —> x temperature —>
Pig eeG conc? M/Iéd , Substrate conc? M/30
Engyme conc? 87x10" Gr per cm.
Fig.8>) En3yme conc? 57x10 yr percm?
En3yme conc? 5x 10 gr per cm?
Salicin Hydrolysis by Enzyme Action. 253
Table VI.
| Salicin hydrolysed per cent. with the following enzyme concentrations
| Temperatures at the per cm.".
beginning and end
of each experiment. | ,
IES <1Oms | 3:8 001 Om | 658'x 10m: || Wt 10s
| | | |
|
15x10-*. | 18x 10-5.
|
|
a
t
=)
30 °2 25 *2 4.6 °8
30 *4-31 °6 —
31 +1
31 °6-31 °7
|
aI
lez
—)
a. ie 1. 98-0 |
wo
a
?
s GW Od us
oa
6) GO
|
aN
=
ee
nS
iw)
61 °3 69-0
—— 60 C7/
is
ive)
a
|
ES
Ne}
KRASERSENS
Turning now to the last stage of the inquiry, the case of the substrate
concentration remaining constant while that of the enzyme changes, it
constitutes the study, properly speaking, of the influence of the enzyme
concentration on the optimum temperature. Although rendered mnmeeeseies
by what precedes, the study is given in order to complete the present
investigation. For this a M/30 dilution of the substrate was chosen and
VOL. LXXXVII.—B. tt
254 Optimum Temperature of Salicin Hydrolysis, etc.
the optimum temperature determined in actions of 15 hours’ duration with
quantities of the enzymic specimen giving concentrations varying between
18x 107° and 18x 107° grm. per cm.?. The numbers obtained are given in
Table VI. The results are recorded graphically in fig. 10.
100. oe op he aos
0!
‘
ai
‘
1
7
‘
(oz)
Salicin hydrolysed (%)—>
Soe Sriok Ss
S
O 80 20 “40 GO Ge
temperature —— >
Substrate conc? M/30.
Fig.lo-{ En3yme conc? |-8x 10*to 18x10 gr per cm
The curves of fig. 10, as well as the M/30 curve of fig. 4 and that of fig. 8,
show that the optimum temperature of the enzyme is the same in each, and,
consequently, independent of the concentration of the enzyme. This holds
true, as shown by two of the curves in fig. 10, even when the proportion
of enzyme to substrate is more than sufficient to produce complete hydrolysis
of the substrate at the optimum point. Here the optimum point is imaginary,
and corresponds to the intersection of the curves representing respectively
the activation and the destruction of the enzyme by heat.
Briefly, then, the outcome of the inquiry is, for an action of known duration,
the optimum temperature of the enzyme investigated is independent alike of
the concentration of the substrate and of the concentration of the enzyme.
Whether the statement be true of enzymes in general—as theoretical con-
siderations would lead one to expect—I propose to answer by fresh experiments
on other types of enzymes.
255
The Resonance of the Tissues as a Factor in the Transmission of
the Pulse and in Blood Pressure.
By Lronarp Hitt, M.B., F.RS., James M. McQueen, M.A., B.Sc, M.B.,
and WILLIAM W. INGRAM, M.B., Ch.B.
(Received June 24,—Read June 26, 1913.)
(From the Physiological Laboratory, London Hospital, and the Pathological Laboratory,
Aberdeen University.)
Systolic blood pressure in man is measured by the pressure indicated on a
manometer scale at the point of disappearance and reappearance of the pulse.
When the pressure is raised in the armlet of the Riva-Rocci or Hill-Barnard,
or their modifications, or in the bag of the pocket sphygmometer (L. Hill), the
pulse is supposed to disappear at the moment when the arterial lumen is
obliterated, and to reappear when the patency of the channel is re-established.
Consequently every effort has been made to secure that the pressure should
be transmitted to the arterial wall as far as possible without loss. Accuracy
in instrumental readings has been held to be conditional on such perfect
transmission of pressure.
Of late years controversy has ranged round the importance of the arterial
wall as a factor in blood pressure, especially in diseased conditions of the
wall, ¢.g. arteriosclerosis. One of us (L. Hill) with Russell Wells (2) and
Martin Flack (3) has shown the importance of the arterial wall in influencing
conduction of the pulse, and has ascribed the high readings obtained in the
arteries of the leg in cases of aortic regurgitation to a better conduction of
the pulse in contracted and more rigid arteries. There remains for us in
this paper to demonstrate another factor, hitherto overlooked, in the taking
of blood-pressure observations, namely, the influence on the arterial pulse
of the resonance of the tissues permeated with arterioles. The pulse is
essentially a phenomenon of periodic vibrations, and by the resonance of the
tissues we denote the property of the tissues to further the pulse vibrations
by synchronous vibrations of like (positive) periodicity.
Our observations have been made in the first place on a man, a boiler-
maker by trade, aged 53 years, whose arteries show on both arms slight
though equal arteriosclerosis. His apex beat is visible within the nipple
line, and his cardiac valves are intact. No aneurismal condition is detected.
His right radial artery pursues an aberrant course, curving some 3 inches
above the styloid process of the radius over the supinator longus muscle on
to the dorsal surface of the forearm, where it runs over the extensor tendons
VOL. LXXXVIL—B, A SONAW SSH!
256 Messrs. L. Hill, J. M. McQueen, and W. W. Ingram.
of the thumb, till it dips between the interossei muscles in the first inter-
osseus space to join the deep carpal arch. The brachial artery in the arm
and the radial artery in the forearm are divided by us into certain positions.
Position I (radial artery) denotes the part of the artery on the back of the
hand that can be covered by the bag of the L. Hill sphygmometer while
space is left for pulse observation distal to it.
Position II (radial artery) denotes that part of the artery which can be
Position 1V
&
covered by the bag as it curves from the dorsal surface to the palmar surface
of the forearm.
Position III (radial artery) denotes the superficial course of the radial
artery in the forearm just previous to its dipping deep between the supinator
longus muscle and the pronator radii teres.
Position IV (brachial artery) denotes that part of the brachial artery that
lies superficially in the antecubital fossa.
In position I the artery lies superficially under the skin, and is placed
upon an unyielding bed of bone, the carpal bones, their ligaments and
the tendons of extensor muscles. Such an observational site may well be
taken as a standard, in the light of which all other positions may be
reviewed. The bag of the pocket syhygmometer applied on the artery at
position I cannot fail to transmit pressure equally to all parts of the artery
The Resonance of the Tissues. 257
beneath the bag, and there can be no loss of pressure here through the faulty
transmission of intervening tissues or through distortion of tissues.
Analysed anatomically position II is similar to position I, while in
position III the radial artery courses over the pronatus quadratus and flexor
loneus pollicis. At position IV the brachial artery lies on the deep tendon
of the brachialis anticus muscle.
Taking readings of disappearance and reappearance of the pulse with the
pocket sphygmometer we find—
mi. of Hg. mm. of Hg.
Position I pulse disappearance 55 pulse reappearance 50
Position II 3 a 55 i Mm 50
Position III M u 130 ~ ig 125
A 105 100
Position IV ¥ ‘ 4 ‘i ES
on ; 75 ‘ ; 70
Substituting a bag of water for one of air the readings are—
mm. of Hg. mm. of Hg,
Position I pulse disappearance 55 pulse reappearance 50
Position II Bs of 45 , u 40
Position III p $3 130 F 43 125
Oi A » ” 1g) » ” 109
Position me t , 65 ; ; 63
The subject was in the horizontal position in all cases.
The low reading of 55-50 mm. of Hg at position I cannot be due to any
fault in the transmission of pressure through the bag to the arterial wall
Consequently, we assume that the pulse has disappeared at 65-50 mm.
of He before the blood flow has ceased through the artery. In other words,
the phenomenon of arresting the pulse by occlusion of the artery is not
brought into play in this observation.
Two methods suggest themselves by means of which it can be proved
that when the pulse ceases to be felt at position I the arterial flow is still
maintained, that the pulse has, as it were, been skimmed off the current.
Keeping the bag of the sphygmometer pressed on position II with a
pressure of 180 mm. of Hg, one can strip the blood out of the artery, and,
to prevent recurrent flow, fix the artery below, as it dips through the
interosseous space. By releasing the pressure at II, the lumen of the empty
artery can be felt to fill with blood when the pressure in the bag registers
115-120 mm. of Hg. It can be felt standing out as a bulging cord at 90 mm.
of Hg, while the pulse returns at 60 mm. of Hg. ;
It is possible to place the armlet so as to cover position III and part of
U 2
258 Messrs. L. Hill, J. M. McQueen, and W. W. Ingram.
II, while the bag is pressed on part of II and part of I, the following reading
can then be taken :—
mim. of Hg.
Pressure an. armlet) fees. .cecna- eee eee ears 90
Pulse disappears below bag at ............ 59
Here there can be no question that the blood flow passes through the
pressure of 90 mm. of Hg, and therefore cannot be arrested by a pressure
of a bag at 55 mm.of Hg. A further possibility suggests itself that the
pulse may be diverted through pressure on the bag, and seek an easier
channel through some branch of the radial artery. Against this supposition
we suggest: first, that the branch chosen must be a big one, otherwise what
the pulse gains in an easier path is lost in the friction due to the narrower
lumen; second, that a pulse would never pass back from the bag at 50 mm.
of Hg, under the armlet at 90 mm. of Hg. Consequently we conclude that
with the bag in position I the pulse is damped down under the bag, while
there is but a trifling obstruction to the blood flow in the artery. The
blood in the artery below the bag takes on the characters of a venous
flow.
The aberrant radial artery where it lies in part of position I, in position II,
and position III was covered by the armlet, and while preventing the
recurrent ulnar pulsation, a reading was taken. The pulse was then found
to disappear and reappear between the limits of 120-130 mm. of Hg. Con-
sequently the aberrant radial artery in positions I and II, overlying bone
ligaments and tendons, can withstand a pressure of, say, 110 mm. of Hg without
the pulse being damped down. But with the bag of the pocket sphygmo-
meter at position I or at position II, the pulse is removed from the blood
current with a pressure of 55-60 mm. of Hg. Yet, according to physical
laws, the pressure is equally delivered to the elastic wall of the artery
by both instruments. The problem is seen then to depend on the air
contained in the armlet in the one case, and on the air contained in the
bag in the other. It is not a matter solely of pressure in the air of the
armlet or of the bag, but the important factor is the state of the air in both
cases as regards periodic vibrations.
The air in the armlet is in a state of periodic vibration. These
vibrations depend on the pulsation of the mass of tissues which surround
the ulna and radius and are embraced by the armlet. At every beat
of the heart the incompressible blood is pumped into the tissues through
arteries large and small, and the pulse of each and every artery is
directed as much outwards into the tissues as inwards upon the blood
stream. Consequently the tissues become a pulsating mass, as can be
The Resonance of the Tissues. 259
registered on a plethysmograph curve. When the bag of the pocket
sphgymometer is applied to the artery, either at position I or at position II,
the pulsations in the air of the bag are at a minimum, because the tissues
lying under the bag are comparatively pulseless. In the case of the armlet,
with its wider embrace of pulsing tissues, the air shows pulsations more
or less synchronous to the pulse in the artery, the arterial pulse is thereby
strengthened and enabled to resist the damping-down effect of the armlet.
Consequently the pressure applied to the arterial wall may be increased from
60 to 100 or 110 mm. of Hg, as the case may be, and yet the pulse persists,
provided the medium through which the pressure is applied is itself in a
condition of like periodic vibration. Of course, the vibrations must be
of such a period as will strengthen the pulse of the artery and not
oppose it.
Tt is on this fundamental experiment that the hypothesis of the resonance
of the tissues is grounded. By this hypothesis we can explain the various
readings obtained by the same instrument (¢.g. bag of pocket syhygmometer)
at positions I, II, If. and IV. Position II is obviously similar to position I.
In position III the radial artery lies as we trace it centrally, first on the
pronator quadratus, and then on the flexor longus pollicis. When the bag is
applied to the artery in position III, there are beneath it fleshy tissues with
numerous arteries in them. Consequently, the tissues below the bag are
throbbing more or less synchronously with the pulse in the radial artery at
position III. The air of the bag is then in a state of periodic vibration, as in
the case of the air of the armlet. Accordingly, the reading becomes the high
one of 130-135 mm. of Hg. The damping-down effect of the bag on the
pulse has been compensated for by the resonance of the tissues beneath it.
Readings with the bag of the pocket sphygmometer placed at position 1V
have been noted to vary from 60 to 100 mm. of Hg. In taking these
readings, the recurrent ulnar pulsation can be damped down at position I,
and the pulse felt at position II. Such variable readings do not oceur
haphazardly; it can be demonstrated that they depend on the varying
anatomical condition of the areas below the bag. Such areas may be
classified into areas of high resonance and areas of low resonance.
If a diagonal line is drawn through the centre of the superficial
brachial artery at position IV (see Diagram), the bag of the sphygmometer
can be so placed that 1/3 of the bag lies to the radial side of the artery and
2/3 on the ulnar side, or, the same length of artery being covered as before
by the bag, 2/3 of the bag can lie to the radial side of the artery and 1/3 to
the ulnar side.
These positions are indicated by the circle (a) and the circle (0).
260 Messrs. L. Hill, J. M. McQueen, and W. W. Ingram.
It is to be noted that the same length of artery is under pressure in both
eases. The bags are covered with the hand in a precisely similar manner,
yet the pulse at a reappears at 90 mm.; at 0, reappears at 60 mm.
This difference can be explained by an analysis of the tissues underlying
the bag in either position. In position a, 2/3 of the bag lies on the fleshy
belly of the supinator longus and biceps, and over the arterial anastomosis of
the radial recurrent artery and the superior profunda artery. In position 8,
2/3 of-the bag lies on the tendinous insertions of the flexor group of
muscles. Here the arterial supply is much less. Consequently, the
resonance of the tissues in position a is greater than the resonance of the
tissues in position 0, and the pulse suffers a great damping-down in
position 0.
Here we have no question of loss of pressure through overlying or
distorting tissues. The tissues over the artery are the same in both cases.
The pressure on each point of the circumference of the bag is the same,
Consequently, it must be that, in position a, the air delivering the pressure
is in a state of greater periodic vibration than the air in the bag in position 0.
The vibrations that underlie the phenomenon of sound are transmitted in
water asin air. We find that when water is substituted in the bag for air
the same results are obtained. The water takes on the periodic vibrations of
the resonating tissues.
L. Hill and Russell Wells (2) have recently shown how important
a factor in the pulse curve is the lability of the arterial wall. It has
also been shown by L. Hill and Martin Flack (3) that, when an artery is
freed from the tissues, and thereby deprived of the support of the tissues
round its wall, the pulse curve is much affected. The lability of the wall is
called into play, and the systolic pressure of the heart is spent in distending
the wall of the artery. It was possible, then, that the artery lying more or
less superficially at positions II, III, and IV, would have its wall distended,
so that the pulse arriving under the bag at position I would be already
damped down before pressure was applied to the artery at position I.
Our experiments show that, at position I, with a pressure, say, of
60 mm. of Hg, the pulse is skimmed off the blood current, but the arterial
flow remains. Consequently, the block on the blood flow is not an
absolute one.
Experiments were made by supporting the superficial artery with the
armlet and with the bag of another sphygmometer, to determine whether
such support played any part in the production of the low pressure reading
at position I.
Our results show that no matter what pressure is raised im the armlet on
positions III and part of II, the pressure in the sphygmometer bag covering
part of position II and part of position I required to obliterate the pulse
On the other hand, when the artery is, in-addition,
supported by varying pressures at position IV, the reading becomes 5-15 mm.
remains the same.
The Resonance of the Tissues.
261
higher. Simultaneous support in positions LV, ill, and part of II, makes the
reading at part of position II and part of position I higher by 5-10 mm.
of Hg.
Table where Supporting Pressure is Applied successively at Elbow and
Forearm.
Supporting pressure of sphygmo-
Supporting pressure in
armlet over part of III
Sphygmometer bag at part of I
|
| meter bag at.elbow, position IV. and part of II. and part of II.
|
mm. of Hg. mm. of Hg mm. of Hg.
Experiment I... 40 40 75 disappearance of pulse.
| 0 0 65 os Fc
Experiment 11... 40 40 73 zs
0) @) 63 x x
Experiment II1... 40 -40 65 » »
0 0 50 » »
Experiment IV ... 0 0) 55 reappearance
8) 40 55 PP 25
40 40 65 a fy
Experiment Y... 0 (@) 55 Cs a
} 0 40 35 » 33
40 40 65 : af
Table where Supporting Pressure is Applied at Forearm only.
Supporting pressure of armlet
mm. of Hg.
0
10
20
30
40
50
60
70
80
90
100
110
in position IIT and part of II.
Sphygmometer bag on part of I
and part of I.
mm. of Hg.
50
50
50
reappearance of pulse.
22
bP]
|
Wote.—It is important to commence from zero and work upwards and not raise the pressure to
110 mm. of Hg all at once, because venous congestion, which is rapidly accommodated for when
rising from zero, otherwise proves a disturbing factor.
262 Messrs. L. Hill, J. M. McQueen, and W. W. Ingram.
Table where Supporting Pressure is Applied as far as possible Simultaneously
at Elbow and Forearm.
Sphygmometer bag at part of
position I and part of IT.
Supporting pressure of armlet Supporting pressure of
applied at Benin IIT and part EPH ee enAD Pe Increase in mm. of Hg on
Sak, ag Sea rare Ny previous reading before
application of supporting pres-
| sure at elbow and forearm.
| mm. of H¢g. mm
Experiment 1... 20 20 5-10
Experiment I1... 30 30 5-10
Experiment III... 40 40 5-10
Experiment [VY ... 50 50 5-10
Note.—Care must be taken |
that the bag at IV is
applied as in Diagram
Tt a.
Yote.—It is not possible to apply these pressures at Positions 1V, III and part of II with
perfect synchronism, as the pressure cannot be raised in the armlet to 50 mm. without two
compressions of the pump.
Accordingly, provided one guards against errors from change in the arterial
wall through manipulation—we have noted that after many readings with the
bag at position I the artery becomes obviously harder and the reading rises—
and, provided one constantly guards against a rise in arterial pressure during
an experiment, then lack of support of the wall may account for a loss of
pressure of 5-10-15 mm. of Hg. But such lack is obviously unable to account
for the low reading at position I of 50-60 mm. of Hg. Further, the experi-
mental observations with the sphyemometer bag on varying positions at IV
show that the main factor must be the resonance of the tissues.
But low readings with the sphygmometer bag are not confined to aberrant
radial arteries. Thus one may observe the same phenomenon on the dorsalis
pedis artery.
Reading with bag on dorsalis pedis horizontal position—
mm. Hg. mm. Hg.
Dorsalis pulse disappears 85. Right radial pulse disappears 145.
3 » reappears 80 ; ne ,, reappears 140.
In this case the dorsalis pedis available was short and the foot was fleshy.
In another case where the dorsalis pedis is longer and the tissues sur-
rounding it scantier, then—
The Resonance of the Tissues. 263
mm. Hg. mm. Hg.
Dorsalis pulse disappears at 55. Left radial pulse disappears at 135.
ss » Yeappears ,, 90 kr . » reappears ,, 130.
Subject in the horizontal posture.
In yet another case the pulse disappeared at 35-40 mm. Hg.
The anterior tibial artery in the leg is overlapped in the upper part of the
leg by the tibialis anticus muscle, in the lower part of the leg by the
extensor longus digitorum, extensor proprius hallucis, and anterior annular
heament. The dorsalis pedis artery is overlapped by the anterior annular
ligament and by the innermost tendon of the extensor brevis digitorum.
Consequently, the artery above the point of application of the sphygmo-
meter bag is well supported. Yet the readings are similar to readings on
positions I and II of the aberrant radial artery.
The low blood-pressure readings obtained with Hill’s pocket sphygmometer
on the aberrant radial artery, or on the dorsalis pedis artery, are due to the
absence of the resonance of the tissues. Provided one could, in the fore-
arm, tie every artery except the radial, and every large branch of the
radial artery, one would find then that the blood-pressure readings taken
by Hill’s pocket sphygmometer, or by the armlet method, would approxi-
mate closely to the low readings found in the aberrant radial artery.
Another method of demonstrating the effect of resonance on the pulse
is the following :—Blood-pressure readings are taken in an individual in
the upright position, from the forearm held at the level of the heart.
The systolic blood-pressure is found to be 120 mm. of Hg (disappearing
pulse index). A similar reading is found in the other arm. One arm is
then fully extended above the head, and the forearm, from the tips of the
fingers to the elbow, is bandaged tightly to render the limb ischemic.
An armlet is fitted to the upper arm, and the pressure is raised
in it well above the systolic pressure to prevent the blood flowing into
the ischemic limb. The bandage is then removed, and the arm lowered
to the heart level. Hill’s pocket sphygmometer is now placed on the
forearm covering the same position as before (the position is previously
outlined with ink) and the radial artery is blocked with one finger to
prevent a pulse from the ulnar recurrent artery ; the pressure in the armlet
is then let down rapidly by pulling the tube off the metal connection of
the compressing bulb. When the first pulses are felt at the wrist, the
bag of the sphygmometer is pressed on to the artery until the pulse is
damped down, a pressure of 70 mm. of He suffices to do this. Soon the
pulse reappears below the bag, and the bag has to be pressed on with, say, a
264 Messrs. L. Hill, J. M. McQueen, and W. W. Ingram.
pressure of 80 mm. of Hg before the pulse again disappears. We find
the systolic blood-pressure readings rise successively from 70 mm. to
80-90-100-110-120-130-140 mm. of Hg. There may, or may not, be a
rebound effect when the blood pressure rises, for a short period, above
what it was at the commencement of the experiment, and above the reading
in the forearm of the other arm.
The ischemic limb on the abolition of pressure in the armlet on the upper
arm is found to gradually swell and becomes red. There is obviously a
marked vaso-dilatation.
Bayliss (4) has shown that when the blood pressure is taken off a limb or an
organ, eg. by blocking the abdominal aorta, an increase in volume of the
limb or organ occurs when the block of the aorta is removed. Bayliss offers
no proof as to which part of the vascular mechanism dilates in this reaction.
A study of the phenomenon in a limb with an aberrant radial artery during
this experiment gives a clue to the vascular conditions present in the reaction.
The aberrant radial artery can be seen to dilate. It stands out like a small
worm on the back of the wrist. The veins on the forearm also dilate. It is
unlikely that the arterioles are constricted when there is visible an increased
blush of the capillary area. We conclude that during Bayliss’ phenomenon,
after a bandage has been used to make the limb ischemic, the main arteries
as well as the arterioles of the limb dilate.
This can be proved by tracings taken with the Dudgeon sphygmograph
from the aberrant radial artery on position I. We use weight extension to
fix the Dudgeon. The base line of the tracings is seen to progressively rise
as the artery dilates. Care must be taken to fix the limb effectively during
this experiment.
A further proof that the main arteries are dilated can be got. by plunging
the congested limb into ice-cold water. After-a period in the cold water the
artery is felt to be very much constricted, and this is confirmed by visual
examination. Massage of the artery brings it back to its original dilated
condition.
A modification of this experiment, viz. releasing the artery and taking the
blood pressure in the ischemic limb as the limb fills with blood, can be
performed. The ischemic limb with an armlet on the upper arm at a
pressure well above the ascertained systolic blood pressure can be plunged
into ice-cold water with ice in it. After a short period the limb, withdrawn
from the ice-cold water, is found to be thoroughly chilled and is dried by
mopping lightly without rubbing. Rubbing might dilate the arteries. When
the pressure is let down suddenly in the armlet, at first the pulse can be
damped down by 40 to 50 mm. of Hg, then the pressure rises, but much more
The Resonance of the Tissues. 265
slowly, to normal or above normal. This is the important point, that the
blood-pressure reading in the radial artery rises much more slowly in the cold
ischemic limb wherein the arteries are constricted than in the warm ischemic
limb wherein the arteries are dilated. At the conclusion of the experiment,
when the blood pressure is back to normal, the aberrant radial artery still
feels like a whipcord—hiehly contracted. Massage of the whipcord artery will
bring it back to the worm-like condition which obtained in the congested
limb.
Accordingly we can conclude that the phenomenon occurs in the dilated or
in the contracted artery—it is immaterial which. Consequently the initial
low blood pressures (as measured by the disappearance of pulse) on allowing
the blood to enter the arteries are independent of the state of the arterial
wall. They are also independent of the peripheral resistance.
Blood-pressure estimations were made on the aberrant radial artery at the
close of these experiments on the warm limb (now congested) and on the
cold limb.
When the systolic pressure had arisen to the normal 120-130 mm. of He
in the forearm in the warm limb the reading obtained at position I on the
dilated aberrant radial artery was 50-60 mm. of Hg. In the cold limb when
the blood-pressure reading in the forearm was found to be 150-160 mm. of
Hg (the same as the initial pressure in the individual tested), the constricted
aberrant radial artery gave a reading of 70-80 mm. of Hg.
We conclude, therefore, that the pulse in either the dilated artery or the
eontracted artery can be damped down by a pressure 70-80 mm. Hg or so
below normal. Experiments similar to the above, and with like result, can
be performed on the dorsalis pedis artery.
We have traced in the ischzemic limb the rise in the size of the beat of the
radial artery, or of the dorsalis pedis artery, or of the aberrant radial artery
(at position I) using both Mackenzie’s polygraph and the weight-extension
method and the Dudgeon sphygmograph, and blocking the artery below to
prevent the recurrent pulse. When the armlet is compressed in the upper
arm and the pressure suddenly let go, one notes that the beat in the congested
limb returns quicker to its normal size than in the ischemic limb. In all
cases the beat takes longer to come to normal when the weight-extension
Dudgeon is used than when the tracings are taken by the polygraph. The
‘weight-extension method of applying the Dudgeon avoids the plethysmo-
graphic effect of the polygraph (Lewis). One often finds the pulse takes
a minute to return to its maximal swing, 2c. until the surrounding tissues are
filled with blood aud resonate with it.
It might be argued in the light of the fact that the return of the maximal
266 Messrs. L. Hill, J. M. McQueen, and W. W. Ingram.
beat is slower in the ischemic limb than in the congested limb, that we
have herein a natural explanation of the initial low pressure readings.
The pulse beats in the ischemic limb are of feeble force, consequently the bag
of the sphygmometer apphed to the artery naturally damps down the feeble
beats. But we have shown that maximal beats, whether the artery is
dilated or contracted, suffer a damping-down in the aberrant radial artery
at extremely low blood pressures. Thus in one experiment, when the
systolic blood pressure was taken at heart level by Hill’s sphygmometer in
the forearm at position III and was found to be 120-130 mm. of Hg, the
returning pulse in the ischemic limb at position III was damped down at
70 mm., and when the blood pressure rose at position III to 120-130 mm.
the pulse in the aberrant radial artery at the back of the wrist at posi-
tion I where maximal beats could be recorded was damped down at
50-60 mm. The feebler pulse beats in the forearm on the radial artery
at position III required 70-80 mm. to damp them down. The maximal
beats on the same radial artery at position I required only 50-60 mm. of
Hg to damp them down. We see, in fact, that the pulse beat, no matter
how forcible, can be damped down by a pressure 70 mm. of Hg or so below
normal blood pressure.
It might be argued that the low blood-pressure readings obtained in this
experiment represent the actual blood pressure in the radial artery, that
there has been a fall of head of pressure as the blood flows into the ischemic
limb. It is not probable that the head of blood pressure would fall greatly,
because the blood flows through the narrow arterioles and still narrower
capillary bed. No matter whether the arterioles and capillary bed are full
or empty, the resistance to the blood stream remains in the friction of the
vessel walls. But blocking the radial artery below the point of measurement
effectively removes the objection that there is a fall of head of pressure.
It might be argued that the fall of pressure continues down the ulnar artery.
But by blocking the radial artery one converts the radial artery into a side
tube measuring lateral pressure from the brachial at the elbow, and the
lateral pressure of the brachial artery at the elbow would not fall. Further,
one can block both radial and ulnar arteries, and the pressure readings taken
from the forearm of the ischemic limb show the same progressive rise. We
conclude that on suddenly lowering the pressure in the armlet the blood
pressure rapidly becomes normal, and the low blood-pressure readings, as
measured by the disappearance of the pulse, are false, both in the ischemic
limb and in the cold ischemic limb.
The explanation of these low blood-pressure readings lies in the diminished
resonance of the empty tissues. The mass of tissue below the bag is not
The Resonance of the Tissues. 267
tense with blood and does not vibrate strongly with the pulse, consequently
the sphygmometer bag acts asa damper. The rise of the pulse to maximal
is aided by the resonance of the tissues. But whether the pulse beat is
maximal or not it is bound to suffer damping down so long as the resonance
of the surrounding tissues is feeble.
It will be noted that the blood pressure in the cold ischemic limb returns
much more slowly to normal than in the warm ischemic limb. Here the
arterioles of the limb are contracted; consequently the blood takes longer
to percolate into the ischemic tissues, the drum-head takes longer to tighten
up, and the resonating effect consequently longer to develop. After dilatation
has been produced in the vessels of a limb, repeatedly made ischemic, it is
less easy to obtain the staircase effect. The bandaging has then to be done
very tightly: on letting go the brachial artery the blood rushes in swiftly,
the skin blushes, and the maximal beat quickly returns.
Many years ago Hiirthle (5) noted that the diastolic pressures taken simul-
taneously with a manometer at the femoral artery and at the carotid artery
were nearly similar, while the systolic pressure at the femoral exceeded that
of the carotid by roughly 68 mm. of Hg. Dawson (6) corroborates this state-
ment, working with the maximum and minimum manometer, but points out
the diastolic pressure in the femoral is always slightly lower than the
diastolic in the carotid.
We would advance the explanation of the higher systolic and lower
diastolic readings in terms of the resonance theory. The abdomen
functionates as a resonator of the pulse, because each organ in it—liver,
spleen, kidney, intestines, ete.—are all pulsating and the cavity is a closed
one. Descent of the diaphragm is compensated for by an outward move-
ment of the abdominal wall. The abdominal wall is an elastic structure.
Consequently the systolic pulse in the aorta and great vessels is surrounded
by more or less synchronous pulsations, which, like the well adjusted tap on
the moving pendulum, augment its swing.
In the case of the higher blood-pressure readings in the leg arteries, com-
pared to the arm readings found by Hill, Flack, Holtzman and Rowlands (1) in
cases of aortic disease, we believe the same resonating effect of the abdominal
cavity is at work, together with the better conduction of the pulse wave
down the tighter abdominal and leg arteries.
It was suggested by one of us (L. Hill) in ‘Further Advances in
Physiology’ (7), that the kidney functionated largely through the mechanism
of the arterial pulse. “In the case of the kidney the blood in the capillary
network, the tissue lymph, and the urine in the tubules are all at one and
the same pressure—the capillary-venous pressure. The whole kidney is
268 The Resonance of the Tissues.
“expanded by each arterial pulse, and drops of urine may be squeezed thereby
into the pelvis from the mouths of the tubules.” Recent work by
R. A. Gesell (8) has shown that the excretion of the urine, the chlorides, urea
and nitrogen is dependent on the arterial pulse. It is to enable the pulse
to be driven to the capillary areas in the kidney or other organ that the
mechanism of a resonation of the tissues is called for. Without some such
mechanism the pulse would be inevitably damped down, especially during
the varying abdominal pressures found with deep inspiration, forced expira-
tion, defeecation, ete.
Further, we would advance the view that by abdominal resonance the
pulse wave is assisted to the most distant peripheral regions of the body.
The aortic pulse finds its way to the tips of the fingers in aortic disease and
to the toes. The longer path is compensated for by abdominal resonance.
Resonation of the tissues must be held to play an important part im the
transmission of the pulse, and thereby to save the work of the heart. The
work of the heart we know is largely conserved by the elastic recoil of the
arteries. But this elastic recoil of the arteries is aided by the resonance of
the tissues. Every artery is in intimate relationship with its immediate
neighbour. The pulse of one individual artery is aided by the pulses of the
other arteries. The vigour of the circulation depends on the tone of the
tissues, on the tautness of skin and muscle, and particularly of the abdominal
wall. The hardened body of the trained athlete swings in full resonance
with the pulse of his heart; the soft, flabby, ill-conditioned body of the
sedentary worker offers a poor slack drum for his heart to thump.
REFERENCES.
Hill, Flack and Holtzman, ‘ Heart,’ 1909, vol. 1, No. 1, p. 76.
Wells, S. Russell, and Hill, Leonard, ‘ Roy. Soc. Proc.,’ 1913, B, vol. 86, pp. 180-186.
Hill and Flack, ‘Roy. Soe. Proe.,’ 1913, B, vol. 86, p. 365.
Bayliss, ‘Journ. Physiol., 1902, vol. 28, p. 220.
Hiirthle, ‘ Arch. f. d. ges. Physiol.,’ vol. 47, p. 32.
Dawson, ‘ Amer. Journ. Physiol.,’ 1905-6, vol. 15, p. 256.
Hill, ‘Further Advances in Physiology,’ London, 1909, p. 153.
Gesell, Robert A., ‘Amer. Journ. Physiol.,’ 1913, vol. 32, No. 1, p. 93.
C9 SU ES Es ES 89 bo
269
On a Method of Studying Transpiration.
By Sir Francis Darwin, F.RS.
(Received October 22,—Read December 4, 1913.)
Transpiration is, perhaps, more directly under the rule of external physical
conditions than any other physiological function. Yet proofs of this
conclusion are wanting, at any rate in regard to the transpiration of leaves.
Thus, as far as I know, we have no complete experimental determination
of the relation between the loss of water-vapour from leaves and the relative
humidity of the air. Nor again have we any complete evidence as to the
effect on transpiration of variation in the illumination to which the leaf is
subjected.
These lacunz in our knowledge depend on the fact that in leaves, tran-
Spiration is largely dependent on the behaviour of the stomata, being
relatively large when they are wide open, and diminishing as they close.
And since the aperture of the stomata depends on external condition, it is
clear that no distinction can be made between the diminution in evaporation
resulting from increased relative humidity of the air, and the diminution
in the transpiration-rate due to stomatal closure. In fact it is impossible to
learn anything accurately concerning transpiration until the varying aperture
of the stoma is excluded from the problem. This might possibly be done by
estimating the transpiration of leaves of aquatic plants in which the stomata
vary but slightly in aperture; but the experiment would not be easily made
in a trustworthy form.
The method I have actually employed is to block the stomata with a fatty
substance,* and then to place the intercellular spaces of the leaves in com-
munication with the external air by means of incisions.
Most of the experiments were made on laurel (P. lawrocerasus). The lower
surface of the leaf was smeared with melted cocoa-butter or with vaseline
rubbed in with the finger, and four to six cuts were made with scissors or a
razor, reaching from the periphery to the midrib between the large veins.
Other plans were also tried, eg. pricking the leaf with a needle or making
numerous small incisions by stabbing with a scalpel.t
The method is similar to that of Stahl,t who showed that greased leaves
pierced with holes assimilate and form starch in the tissues surrounding
* Cocoa butter in the earlier experiments, vaseline in all the later ones,
+ The method was described in a paper read at Section K of the Sheffield meeting of
the British Association, 1910 (title-alone published).
t ‘Bot. Zeit.,’ 1894.
270 Sir F. Darwin.
the wounds, whereas greased leaves without such artificial stomata formed
none or hardly any.
It may be objected that the stomata are not completely or uniformly
closed by greasing, that some remain open, and that it is to the opening and
closing of these in hight and darkness that the rise and fall of the transpira-
tion of the incised leaves is due. I find it difficult to believe that the
general objection here discussed is sound, because experiments with the
_ porometer* have convinced me that even a careless application of vaseline
absolutely closes the stomata. It may be urged that in Experiment LO 2
(p. 271) the effect of grease is only to reduce transpiration from 379 to
10-9, we, from 100 to 2°88. It must be remembered, however, that fatty
substances are not impermeable to water, and that at any rate part of the
2°88 per cent. must be due to cuticular transpiration.
Another source of error should be guarded against. Mr. Blackman} has
shown that a process of healing occurs in wounded laurel leaves. The
beginning of the process is, however, marked by the edges of the wounds
becoming translucent. As soon as this oceurs the specimen should be dis-
carded or fresh incisions made.
The following experiment, LO 2, October 2, 1912, gives an idea of the
effect of greasing and slitting. It seems clear that the result is com-
parable (as far as magnitude is concerned) with normal stomatal transpira-
tion :—
Experiment LO 2. October 2,1912. P. lawrocerasus.
A laurel branch cut under water with 10 leaves (one being small) having
a stoma-bearing area of 600 cm.?.
Fitted to a potometer (diameter of tube 0°95 mm.). At a north window,
where the temperature during the observations varied between 13°6° and
15:2° C. and the relative humidity between 59 and 69 per cent. In the
following abbreviated record of the experiment the potometer readings
are corrected for differences in relative humidity.
A.M. Transpiration.
11.10 439t
27 427
53 379
P.M.
12.18 Finished vaselining leaves on both surfaces.
* See F. Darwin and D. F. M. Pertz, ‘Roy. Soc. Proc.’ 1911, B, vol. 84, p. 137, for a
description of the porometer.
+ F. Blackman and G. Mattheei, ‘ Annals of Botany,’ 1901, vol. 16.
{ The figure 439 is obtained from the number of seconds (viz. 22°8) in which the
column of water in the potometer tube travels 1 cm., which means the absorption of
On a Method of Studying Transpiration. 271
P.M. Transpiration.
12.20 285
ot 64:7
43 Surface of branch vaselined.
59 A473
4.36 181
Oct. 3—
A.M.
10.23 Fresh surface cut to branch.
ete 10°9
32 Four incisions made per leaf, 7.e. two on each side of midrib.
36 943
P.M.
12.5 One more slit per side.
(i 196
12 One more slit, making four per side
44 255
Oct. 4—
A.M.
10.11 Fresh surface cut to branch.
1G155) 234
Tt will be seen that the coating of vaseline on the leaves and surface of
the branch does not completely check transpiration. Thus, as above
mentioned, on the second day (October 3), when the original negative
pressure must have disappeared, transpiration had only been reduced from
379 to 10°9 or from 100 to 2°88. This fact is in the present instance of
little importance, as my object is to illustrate the effect of incisions on
the transpiration rate.
It is obvious (i) that when the lamina is cut into strips the transpiration
rises with great rapidity; (ii) that although in this instance it does not
obtain the rate of transpiration observed when the stomata were open, the
two are comparable for practical purposes.
In the case of these slit leaves it is of some interest to know the amount
of connection between the external air and the intercellular spaces. This
was estimated from the observations on the laurel twig (Experiment LO 2)
just described. Each leaf had eight incisions (four per side), varying in
0:00708 c.c. The figure 439 is the reciprocal of 22°8 multiplied by 10,000. To convert
the number 439 into cubic centimetre it is only necessary to multiply it by 2°55 mm.?
which gives the rate, in this case 1°12 ¢.c, per hour per 600 cm.? or 18°7 c.c. per square
metre of stoma-bearing area.
VOL, LXXXVII.—B. x
272 Sir F. Darwin.
length from about 25 to 40 mm. The sum of the lengths of the incisions
= 2437 mm. The thickness of the leaves was taken as 0°38 mm., and since
each incision exposes two leaf-sections to the air, the total area of section
exposed by the experiment is
2 x 2437 mm. x 0°38 mm. = 1852 mm.? = 18°52 em.?.
The stoma-bearing area of the 10 leaves, omitting the mid-ribs, was
600 cm.?, so that the amount of surface exposed by incision is 1852 per
600 or 3:09 per cent. Unger* gives for P. lawrocerasus the intercellular
spaces as 21:9, say, 22 per cent.t of the volume of the leaf. Therefore of the
transverse section exposed by incision only 22 per cent. is intercellular space.
We may therefore say that in a laurel leaf having four incisions on each
half of the lamina the transpiratory apertures connecting the intercellular
spaces with outer air are 22 x 309/100 or 0°68 per cent. of the area of the
leaf. Since these correspond in function to stomata it is worth while
comparing them with actual stomata.
A rough calculation gave the area of the laurel stomata as 0°88 per cent. of
that of the leaf. The transpiring area of the slit leaves is, therefore, much
the same as that of the stomatal apertures under ordinary conditions.
The Effect of Changes in the Humidity of the Air.
The method of incision has been used in studying the effects, on transpira-
tion, of variations in the relative humidity of the air; and this has led to a
rough plan for reducing transpirations at varying humidities to a common
standard. The method of producing a damp atmosphere was a simple one.
At first the plant was covered with a large bell-jar resting on a ground-
glass plate, and so arranged that a current of air, dry or moist, could be
drawn through it. But finally I came to the conclusion that a simpler
method was preferable, namely, to change the relative humidity by raising
or lowering the bell-jar; in this way—assuming that the laboratory air is
fairly dry—it is easy to change the relative humidity from 50 per cent.
to 95 per cent., which is sufficient for my purposet The wet and dry
* ©Sitzb. K. Akad. Wien,’ 1854, vol. 12, p. 367.
+ Microscopic examination of a transverse section led me to estimate the air spaces as
roughly 25 per cent.
t It is unfortunate that these observations, with the exception of Experiment 8, were
not made in darkness or in constant light. The experiments which are most likely to be
vitiated by this fault are Nos. 3, 4,and 7. Experiment 4 might be expected to give an
especially bad result from the effect of dull light at the end of the experiment. But the
diagram, fig. 4, shows rather striking uniformity in the relation between transpiration
and humidity of air. In Experiment 3 the diagram is not very satisfactory in any case,
but omission of the last two readings (the ones under suspicion) would not alter the
On a Method of Studying Transpiration. 273
bulb thermometers were in the upper part of the jar, while the branch
had leaves in both lower and upper regions. I did not find this to be a
serious source of error, and it is one which might be avoided by fitting
an apparatus by which the air in the bell jar could be stirred and thoroughly
mixed, as indeed was done in some of the later experiments.
The rate of transpiration was estimated by a potometer, not one of the
type formerly used by me,* in which an air bubble is timed as it passes
rapidly along a narrow capillary tube, but one in which the free end of
the water-column is timed with a stop-watch as it passes along a horizontal
tube of about a millimetre internal diameter.t It is, in fact, like Kohl’s
potometer, or that figured in Pfeffer’s ‘Physiology, though the method of
bringing the column back to zero is not identical with either. I have
not thought it necessary to give the actual quantities of water absorbed
by the plant per hour, but merely a series of numbers proportional to the
rate of absorption.
In all experiments (except No. 8) the plants were placed close to the north
windows of the laboratory; the action of the stomata was in all cases
excluded by a coating of grease, transpiration taking place only by incisions,
as above described.
In the following tables T means temperature, wy stands for relative
humidity :—
Experiment 1—November 6, 1909. P. lawrocerasus. Fig. 1. Cut branch in
potometer.
Time. Period. Rate. | At, w.
| mae per cent.
10.13 A.M. i Bou aaualors 74,
WOSE) il 38 elisi6 74
10.49 ,, iii 36. =|) 2 3N6 74
Bell-jar over plant.
MUNG 5, iv | 16 | en 91
11.28 ,, v 20 anf 92
ie vi 14) 7s) R50 93
Wes pe vii 17 15 °2 94,
|
transpiration curve. Experiment 7, in which the last reading was taken at sunset, gives,
nevertheless, a good straight diagonal, as seen in fig. 7. A number of experiments were
made (like Experiment 8) in the dark room. I cannot see that they differ as a whole
from those illustrated in the present paper.
* BF. Darwin and R. Phillips, ‘Camb, Phil. Soc.,’ 1886, vol. 5; see also F. Darwin and
Acton, ‘Physiology of Plants, 1901, 3rd Edit., p. 79.
t+ In all the later experiments the diameter was either 0°95 mm. or 1‘1 mm.
x Y
Pape Sir F. Darwin.
40
30
20
110\7 100. G90) E i ieOsiea7Oemmeo
Fie. 1 (Experiment 1).
In the figures the ordinates represent transpiration rates, while the relative
humidity (y) is given on the horizontal axis. Thus if transpiration varies
directly as the relative humidity, the diagram should give a straight diagonal
line. The fact that the diagonal does not pass through the intersection of
the axes will be discussed later.
Experiment 2.—November 8, 1909. P. lauwrocerasus. Fig. 2.
Time. Period. Rate. 40, w.
| '
| °C. | percent.
11.48 A.M. i 56 Iastey | 53
12.3 P.M. | ii | 56 15 *4 57
| Bell-jar over plant.
12.24 ,, | il 35 15°1 89
12.35 ,, iv 32 15 °2 92
12.44 ,, | v 27 | 15-4 93
25s vi 17 15°6 94
|
i |
110 100 90 80 70 60 50
Fie. 2 (Experiment 2).
It will be seen that the dots representing transpiration for various values
of y are by no means in a straight line. This I take to be “lag,” that
On a Method of Studying Transpiration. 275
is to say, a relatively slow response to the change in the humidity of the
air (yr). When the air is drying instead of becoming damper the “lag” is of
the opposite character, as seen in fig. 6.
Experiment 3.—November 15,1909. P. laurocerasus. Fig. 3.
Time. Period. Rate. | T. v.
sane 7 ie
| me: per cent.
ee i 57-0 15-4 54
Ze 2 Bell over plant. .
FE 92) eu Taare TO 30 le ae
nee fee 235 | 15°4 83
3.28 ,, | Water poured on floor of bell.
bimeres 7 } vy | 18-0 | 155 94
estore tee } vi 110 15-4 94
) |
10 100 90 80 70 60 °° 50
Fic. 3 (Experiment 3).
Experiment 4.—January 1,1910. P. laurocerasus. Fig. 4.
| |
| Time. : Period. Rate. | iT ry.
|
“Che SNP percent,
12.35 P.M i le RAD TAA | 3
IPAS | Bell-jar on.
12.59 ,, | i 37 °9 14 °6 63
2.30 ,, iii 333 14:2 61
orlaias, iv 30 °7 14°7 68 |
3.22 ,, v 25°8 14°8 72
3.35 ,, Vi 22°6 14°7 77
BEC ter} vii 19-2 14°7 81
44 ,, Vili 16:1 149 | 89
276 Sir F. Darwin.
100) 190 580 705 60) 50
Fie. 4 (Experiment 4).
Experiment 5.—January 3, 1910. P. lawrocerasus. Fig. 5.
|
Time. Period. Rate. As W.
|
| fy =
mice per cent.
10.32 4.M. i 39 “4 16 °3 61
OSes ii 29 °6 15°8 72
IDUBY) 5, | iii 28-1 16-0 74
12.19 P.M. iv 22°3 16 4 78
WAS Vv 21°3 16 °4 82
1 TAB. 6 vi 17°2 16 “4 87
| 113333, vii 16°5 16:0 85
| LAO), viii 14°3 16 ‘1 89
1.54 ,, ix 10 °6 16°1 94
|
40
.
a
0:
4 |
100 90 80 70 60
Fie. 5 (Experiment 5).
During the above observations the bell-jar had been gradually lowered, 1.2.
the supports replaced by smaller ones until only a crack, 1 or 2 mm. in height,
remained. The bell was now (1.57) raised to 7 mm. and a current of air
drawn through. The supports were gradually increased in height and finally
(3.20) the bell was removed altogether.
On a Method of Studying Transpiration. 277
Experiment 6 (= 5 continued).—Fig. 6.
| Time. Period. Rate. AL wes
| se: per cent. |
| 1.54 Pat i 10°6 16°1 94 |
} 2.24 ,, li 20 °8 16°3 | 78 |
2.39 5; ii 24°7 16°3 72
36) ee iv 30°38 16 °5 69
Bai hee y 34-6 162 6rd
o29) 3, vi 41°7 L770 59 |
3.33, Vil 43 5 17:0 58 |
LOOfr SORURIAOLs “70%. “co 50
Fic. 6 (Experiment 6).
In Experiment 6 the air is drying instead of becoming damper, and the
“lag” is of an opposite character to that in fig. 2.
Experiment 7—November 15,1909. P. /aurocerasus. Fig. 7.
Time. Period. —— Rate. lb w.
}
Se per cent. |
| mo mee } i 57 ‘0 | Ate | 54
12.12 p.m. Bell on.
LP ; ii | 50-0 154 | 60
2.54 ,, oa | ae i |
319 » } ili |} 23°5 15°4 | 83
S250 Water on floor of bell. ;
oe see a le | 130 155 | 94
Lee : } Teenie ahs 0 | Tene at 2
Bell removed.
ane 5 } vi 54-08 | 15-9 50
|
* The value of this observation is doubtful; it is marked with a x in fig. 7 and omitted
drawing the diagonal.
278 Sir F. Darwin.
110 100 90 80 70 60 650
Fie. 7 (Experiment 7).
Experiment 8.—April 22,1912. P. lawrocerasus. Fig. 8. Apparatus fitted
up in the Dark Room.
Time. Period. | Rate. ote w.
l |
Ce per cent.
10.45 A.M. i | 22 °4 17 °3 60
to 11.30 _,, | (average)
TES 2 aes | Bell jar over plantisupported on blocks 25 mm. high.
1156n0e ii } 1571 17:8 | 74
128) | Blocks reduced to 2 mm.
12.14 pm. | iii (eR ETO? HAL -o 77
152480 ee iv FOZ Weel S10 84
19:89, ae v | 8-0 18°1 88
NAS). v1 7°8 | Ueyaal 88
12.44 , |. vii | 6°5 18-1 91
12.54 ,, | Vili | 5°3 18 -2 93
12.59 ,, ix | 50 | 1872 94
ZolOws x 3°5 18 -2 97
2.29 | xi | 3°3 18 °3 98
20
Fia. 8 (Experiment 8).
The general characteristics of the illustrations above given are
(1) The points which represent the transpiration for different degrees of
relative humidity are roughly in a straight line—from which it follows that
a definite relation of some sort exists between transpiration and relative
On a Method of Studying Transpiration. 279
humidity. This conclusion, which is a physical necessity, does not seem to
have been definitely proved or represented diagrammatically.
In some cases (¢.g. figs. 2 and 6) the line of dots (~e. the transpiration
curve) is not straight—the change in rate of transpiration lags behind the
change in y—for reasons not yet clear.
(2) The second characteristic of the diagrams is that the diagonal does
not pass through the point of intersection of the axes—or, in other words,
transpiration is not zero in saturated air. I have not hitherto seen this
graphically represented as the result of experiment, although it might have
been foretold. The fact that transpiration occurs in saturated air, and that
it is due to the production of heat in plant-respiration was first made clear
by Sachs*, who proposed that the fact should be utilised as a means of
measuring the “Higenwirme” of plants.j| We shall see later that the
diagram (fig. 9) may perhaps be applied to the same end. ‘The position of
30
iS)
fo}
Transpiration.
B
Relative Humidity.
Fia. 9.
the point G varies in different cases. In the earlier experiments, I estimated
AG = 7, but I now consider 5 a more reasonable average. The construction
here given has been used throughout my work for the rough reduction of
transpiration-rates to a common degree of relative humidity. Thus,
supposing that in fig. 9 the transpiration-rates DC and EB have been
obtained under different conditions of illumination, it is clear that we
cannot estimate the effect of such conditions until the amounts have been
corrected for the differences in relative humidity.
* ‘Sitzb. K. Akad. Wien,’ 1857, vol. 26, p. 326.
+ See Sachs, ‘ Physiologie Expérimentale,’ 1868, p. 249 (the French translation of his
book on plant physiology).
280 On a Method of Studying Transpiration.
Now
il) Cree, LCG i _ 105—70 _ 35
EB BG DC = 5G x EB. ah DO = Fosrs0 * EB = op BB:
We will suppose that in an experiment on the effect of illumination we
find the transpiration-rate in the light (relative humidity 70 per cent.) to
be 120; while the rate in the dark (humidity 80 per cent.) is 75. We must
multiply 75 by 35/25. The product 105 is the transpiration in the dark
room (humidity 80 per cent.) reduced to humidity 70 per cent., and there-
fore now comparable with transpiration in the light, z.e. 120. Thus
Transpiration in light _ 120 _ 114
Transpiration in dark 105 100°
Sir Joseph Larmor has been good enough to point out to me that it is
possible to get a rough idea of the temperature of the leaf at full saturation,
i.e. of the leaf temperature which in fig. 9 produces the amount of transpira-
tion (or what may be perhaps called distillation) equal to AF. The oblique
line, or curve of transpiration, cuts the horizontal at 105, 2.e. at 5 per cent.
above saturation. The figure shows that, in supersaturated air, i.e. 5 per
cent. above saturation, transpiration is nil. The hypothetical degree of
supersaturation should be a measure of the transpiration AF at the satura-
tion point, and therefore of the internal temperature which can distil off
water in saturated air. Assumine* the temperature of the air to be 16° C.,
the vapour pressure would be 13°51. If we add 5 per cent. to this we get
14-2, which is the vapour pressure corresponding to 16°8°, or 0°8° C. above
the temperature of the air. There seems no improbability in leaf-respiration
producing, under the conditions of the experiment, a temperature of roughly
1° C. above that of the atmosphere. In my earlier experiments I concluded
that the transpiration curve DEFG cut the horizontal at 107. This would
have given a temperature 1:1° C. above that of the air, instead of 0:8° C.
It should be noted that the distance AF, z.c. the amount of transpiration in
saturated air, will depend on the general temperature, since respiration is
greatly influenced by temperature. We have some evidence on this point,
but the experiment needs careful repetition.
It is remarkable that, as far as I know, the method here used for plotting
the relation between transpiration and relative humidity has not been
employed. If Le Cleret had treated his results in this way, he might,
perhaps, have obtained a result like mine. —
I cannot conclude without expressing my indebtedness to Miss D. F. Pertz
for much kind help in the laboratory.
* The figure is a diagram not taken from any one experiment.
+ Le Clere, ‘ Ann. Sci. Nat.,’ 1883, vol. 16.
281
The Effect of Inght on the Transpiration of Leaves.*
By Sir Francis Darwin, F.RBS.
(Received October 22,Read December 4, 1913.)
The method employed is essentially that described in my papert “On a
Method of Studying Transpiration,” where it was applied to the investigation
of the relation between the relative humidity of the air and the loss of water
by leaves. The stomata of the plants used were closed by vaseline or cocoa-fat
rubbed in, and the leaves were then incised to allow of transpiration. No
attempt was made to subject the plants to light of known intensity. My
object was to compare the transpiration occurring in a dark room with that
in a north light at a laboratory window. The rates of transpiration were
estimated either by weighing or by means of a potometer, and the general
plan was to subject the plant to alternate light and dark periods of something
like an hour.; The psychrometric condition of the laboratory air and that of
the dark room was estimated by the wet and dry bulb thermometer, and the
transpiration rates corrected for any differences, in the manner described in
the paper above referred to.
The first experiment was made by a plan which has some merits, but was
afterwards replaced by the simpler method of moving the apparatus from the
window to dark room and back again to the light.
Experiment 1—December 9, 1909. P. lawrocerasus.
Branch fitted to potometer December 8 and the lower surfaces of the leaves
greased ; leaves cut about 10 a.m., December 9.
Placed under a bell-jar through which a current of laboratory air is drawn
* Jt is not easy to find any recorded experiments on the transpiration of leaves in
light and darkness, in which the action of the stomata is absolutely excluded. In
Bonnier and Mangin’s experiments on the transpiration of fungi this is 7pso facto the
case (see ‘Ann. Sc. Nat.,’ 1884, vol. 17, p. 298). The average of the experiments on
Trametes suaveolens is:—L/D = 119/100. For Polyporus versicolor the corresponding
fraction is 127/100. The symbol L/D stands for the relation between the transpiration
in light and darkness.
+ ‘Roy. Soc. Proc.,’ this vol., p. 269.
t A few weighing experiments were, however, made on the effect of the natural
darkening occurring at night. The average of eight experiments gave the proportion
between transpiration in the day (L) and in the evening (D), as L/D = 129/100.
Four experiments made with the potometer under similar conditions gave
day (L)/evening (D) = 112/100. This subject, including the effect of continuous dark-
ness, requires fresh investigation.
282 Sir F. Darwin.
to keep the relative humidity (yy) as constant as possible. The rates of trans-
piration are given as corrected. North light.
: |
Time. Rate corrected. | 1s v.
| 7 (Of per cent.
10.42 A.M. 24 °8 14 °2 65
10.54 ,, 24°7 14 °2 64
hee 2 } Covered bell-jar with a black bag.
Thien 27-1 l 14°6 62 |
12.5 P.M. BPACIL | 14°6 | 63 |
12.30 ,, Light : cloth bag removed.
12.41 ,, 24-9 | 15 °2 62
2.46 ,, 25-8 157 63
a0). 28 -0 15-7 63
2s Dark : cloth bag replaced.
3.43. 250 15 °9 63
3.57 ,, | 25 -4 15°8 63
Ash. 260 — —_—
esult.—The fall in transpiration-rate between 11.21 a.m. and 12.5 P.M. is
27-1 to 22:1 or 123/100. The rise in the next period is from 2271 to 28-0 or
100/127 ; the diminution in the final dark period is 108/100. The average
proportion between the transpiration in light and darkness (L/D) is
119/100.
Experiment 2.—April 11,1911. P. laurocerasus.
Potometer: T 15:°0-16:2° C. +f 46-56 per cent. Transpiration corrected.
Time. | Rate. Time. ' Rate.
11.17 a.m. | 164 11.55 a.M. 107
HLS | | 140 158 2 103
TMS Ye oy, 122 12.13 P.M. | 108
OVA | 116 12:28 ,, 95
In dark room. 4.10 ,, 109
TiS 115 tea | 110
|
Transpiration was falling (in the light) from 11.17 to 11.41; the effect of
darkness was to diminish rather than to increase the rate of fall. The total
change in the dark is a fall from 115 to 110, or L/D = 105/100.
Potometer: leaves slit at 10.05 a.m.
Experiment 3.—April 19, 1911.
T 16:1-17-:0° C. > 50-62 per cent.
Transpiration corrected.
The Effect of Light on the Transpiration of Leaves.
P. lauwrocerasus.
|
Time. Rate. Time. Rate.
11.22 a.m. 159 11.55 A.M. 186
THB) 5 149 12.8 P.M. 168
11.45 __,, 153 3:8). 187
IDLO)! In dark room SHA 5 181
The transpiration had been steady for some time before the plant was
placed in the dark room. The only clear effect was a rise in transpiration-
rate from 153 to 181, or L/D = 100/118.
Experiment 4—April 20,1911. P. lawrocerasus.
Potometer: leaves greased and slit 10.30 a.m. T15-0-16:6°C. 44-47 per
cent.
Time. Rate. Time. Rate.
10.40 A.M. 757 12.2 P.M. 797
IO) 814 12.43 ,, 610
Dark room. 12.45 ,, Light.
Taco; 902 12.48 ,, 493
11.24 ,, 897 12156) 5; 550
iB 5, 816 3.30 ,, 559
In this experiment the effect of the dark room is doubtful, as the rate was
not steady before darkness. If we assume that the fall in rate was due to
darkness, we have the big effect of fall from 902 to 493 or 183/100. The
subsequent rise in the light is from 493 to 559 or D/L = 100/113.
The average of the light and dark effects is L/D = 148/100.
P. laurocerasus.
Experiment 5.—April 22, 1911.
Potometer: leaves greased and slit 10.31 a.m.
52 per cent.
Tey 1G Geoienoney ev) Ale
Time. Rate. Time. Rate.
10.47 A.M. 210 11.54 a.m. 157
IN@){50) 233 12.5 P.M. 162
10.52 ,, 208 TIPS) 154
10.58 ,, 213 22 162
TUL 5, In dark room WASP) 171
LIST, 12)39e 174
GAN ee 176 WAGO) 5, 174
D48: 5; Tn light
284 Sir F. Darwin.
Transpiration was approximately steady before darkening and fell from 213
to 175, or from 121 to 100, during actual darkness; or, if we include the
reading taken at 11.54, it fell from 213 to 157, or from 136 to 100.
There is the same doubt about the effect of subsequent illumination. If
we compare the end of the dark period with the last reading taken in the
light the effect is nil. If we compare reading at the beginning of the light
(157) with that at the end (174) we get a rise of 100 to 111.
On the whole it is fairest to take the darkening effect as 136: 100, the light
as 100:111. The average of the light and dark effects is L/D = 124/100.
Experiment 6.—November 15-16, 1911. LP. laurocerasus. Potometer.
Time. Rate. | Time. Rate.
|
Noy. 15. 11.30 a.m. Light—at east window.
10.25 a.M. Leaves slit. 11.40 .,, 146
TIS} 65 In dark room. IRS Aver 134
Nov. 16. 35 5 12.1 P.M. 137
10.17 a.m. Cut fresh surface to branch. leaeZ on 143
Os, 119 12.40 5, 148
lie 5 120 12.50 ,, 162
Dy 5 126 12.57. ,, 153
The effect of light may be taken as increasing the rate from 126 to 158
(the average of last two readings), or L/D = 125/100.
Experiment 7.—November 17,1911. LP. laurocerasus. Potometer.
Time. Rate. Time. Rate.
10.15 a.m. Leaves cut. 11.16 a.m. In dark room.
ION 192 11.28 ,, 317
10.34 ,, 316 11.40 ,, 292
NOB) op 325 12.0 Noon 294
10.50 ,, 300. 12.37 P.M. 278
Te 5, 330 WBS oy 286
TIALS), 308 12.56 ,, 278
Shortly before the period of darkness the rate may be taken as = 320
‘(average of last two readings), at the end of the dark period it is 280
(average as above); this gives a diminution in transpiration equal 114 to 100,
or L/D = 114/100.
The result of the above series is given in the following table; L—D means
that darkness followed light, D—L indicating the opposite. The last column
gives the effect as a percentage. Where, as on April 20 and April 22, there
is a L—D as well as a D— effect, the average is given :—
The Effect of Inght on the Transpiration of Leaves. 285
| i
Experiment. | Date: Effect of light or of |
dark.
|
|
|
|
|
|
|
|
|
|
1 Dee, O80) 1 esi Mee ren) |
| D—L 100 — 127 } 19 per cent.
L—D_ | 108 — 100 |
2 Ase, Ul, 160 Al) Thy CR 5 OO | BS
3 Msesite) WD | MOOm- TOON On a
4 , 20, IGM 2A) ees) eR (0s
D—L | 100: 118 »
5 SPL TOI SA esa) TIES poe
| D—L 100 : 110 22 |
6 Novels (ote te! = 1)" |) 100 =) Tase25) |
7 -, Al, TON el) ose) NT 2 aie) Fey
Average ...... L/D = 119/100.
In some cases transpiration is but slightly affected by darkness, as in the
ollowing experiments.
The material was supplied by small branches of laurel (P. lawrocerasus),
having, as a rule, four leaves, vaselined and cut (four incisions per leaf) in
the usual way. A branch was fitted to a simple form of potometer consisting
of a pipette graduated to 0:01 cc. The pipette was fixed vertically and the
branch attached to the lower end by rubber tube; as the plant absorbs
water the descent of the meniscus is read with a lens, by which means
errors of parallax are fairly well avoided.
The experiments were made alternately in a dark room and at the north or
east window of the laboratory.* Readings were generally continued for an
hour before the change from light to darkness, or vice versé, was made. The
results, u.e. the amounts of water absorbed per hour in light and darkness
were corrected for psychometric differences. The dates of the experiments
summarised below were April 21, 22, 23, 28, 29, May 1, 2, 3, 1913.
The results were somewhat irregular and are therefore given in the form of
an average. A single experiment is, however, given in detail.
* Jn a few cases in a dark room which could be illuminated by opening the shutter.
The room was to the south and care was taken to avoid sunshine.
286
Sir F. Darwin.
Experiment 8.—April 28, 1913. P. lawrocerasus.
Leaves, five in number, vaselined and cut into strips at 9.40 a.m.
potometer, 60 cm. from window of dark room, shutter open.
vv 63-65 per cent. Transpiration corrected for y. Dull day.
|
[
Time. Reading. Rates per hour.
C.c. c.¢
9.51 A.M. 0-080
1005555; 0-103 0°156
LOWSs 55 0-142 0152
10.34 ,, 0-190 0°151
10.45, | 0217 0:147
1O50RR | Shutters closed. Dark.
10.50 ,, 0 °233 0-192
WO oy 0 °254 0°126
TOL Hs) 5 0-292 0'152
TI SOMEs. 0 "326 0°136
11.45 ,, 0 “361 . 0°140
| Shutters opened. Light.
12.0 NOON 0 394 0°182
12.15 P.M. 0-430 0144
12.30 ,, 0-463 0 °132
12.45 ,, 0-497 0°136
12.46 ,, Shutters closed. Dark.
2.30 ,, 0-715 0-125
Vertical
1h 5 oeD.
I have usually estimated the transpiration by taking the average of the
two last readings in each period, Light (L) or Dark (D), as the case may be.
But in Experiment 8 the first L reading should clearly be the average of
the last L and the first D reading, zc. 170. The other averages are D 138,
L 134, D 125; they are included in the general average.
The results of the above-named eight experiments show considerable
irregularity and no clear impression is gained by inspection.
taken the average of 31 readings from the series, 18 representing transpira-
tion in light, and 13 in dark. They are as follows :—
I have therefore
Light. Dark. Light. Dark
153 155 134. | 125
214 200 | 157
186 183 161 141
216 224 181
170 166 212 152
130 122 170 138
120 149
96 118 110 100
170 138 104
Average 162 : 144 ]
or
113 : 100 f
L/D = 113/100.
The Effect of Laght on the Transpiration of Leaves. 287
Another series of similar experiments was made by Miss Pertz on
P. lawrocerasus, using a Ganong potometer.*
The following example shows a definite light and dark effect, in spite of a
good deal of irregularity. The figures are corrected for relative humidity,
which varies between 56 and 60 per cent., while the temperature lay between.
15°5° and 16:1° C.
Experiment 9.—May 3, 1913.
|| |
Time. Transpiration. | » Average. | Time. Transpiration. | Average.
|
woh aN =
Light.
10.49 a.m. 150 12.0 Noon Light.
TLCS aie 168 | 12.5 P.M. 157
neeslileD 1 ( 167 TAO 25 144.
PETE G: 164 \ He ION 133
1135) ,, 176 12.20: ,, 142 ue
Darkness. 12.35 ,, 156
11.43 ,, 172
11.50 _,, 139
11.59 ;, 137 f| 188
In the third column is given the average of the last two readings in the’
three periods Light, Dark, Light; thus the result of the experiment of
May 3 is L170, D 138, L149.
The following table gives the results of the series, May 3 to May 12,1913,
Experiments 9 to 15 :— ;
Date. Expt. L. D. L.
1913.
Mayona onic: 9 170 138 149
56) De raaatomes 10 110 100 104,
ae Gc on 11 134 107 134
CWE area a 12 144 121 132
Rens ratte taes 13 113 109 103
ee LO eames 14 70 65 67
ip UZ cosooosneoce 15 17 60 67
Sumagear 818 | oo 756
|
OPES seasoe 117 LOO LOS
Or taking the average of the two light readings, we have—
L/D = 113/100
* “Plant Physiology, by W. F. Ganong, 1908. The instrument is supplied by
Messrs. Bausch and Lomb.
VOL. LXXXVII.—B. Y
288 Sir F. Darwin.
The following experiments were made in an improvised laboratory having
a bright north light; but the dark room was not perfect and the plants
could not be exposed to absolute darkness. The potometer used was of the
Kohl type, having a horizontal tube 0°95 mm. internal diameter. The march
of the meniscus was generally timed over half a centimetre. The meniscus
is brought back to zero by turning a tap and allowing water to enter, as in
the instrument designed by Prof. Ganong.
The rate of transpiration is corrected for y (relative humidity).
Experiment 16.—May 7, 1913. P. lawrocerasus.
May 6.—Branch, with seven last year’s leaves, gathered at night.
May 7, 10 a.m.—Finished vaselining and making incisions in the leaves.
Fresh surface made to branch; apparatus at an east window; dull morning.
yw between 65 and 82 per cent.
T 12°7-16°8° C.
For each period, light or dark, the average rate of transpiration is given.
Time. Rate. | Time. Rate.
Light. | 12.18 pia. | Dark.
10.20 A.M. - VAT, os,
10.40 ,, Bee i ae 187
10.47 ,, Dark. | Heo ies Light.
| THLIO 5, BING cp
Piney 2) eatincoel | 255
Fi. 19" 8 | Light. | 6.53, 195
iLO) 5, GAD dull light.
| 12.9 P.M. 277 ¥
i
Omitting the last reading (as giving the effect of dull light), we have the
average rates: Light = 278, Dark 208, or L/D = 134/100.
Experiment 17.—May 12,1913. P. laurocerasus.
9.45 A.M.—Cut fresh surface to a branch which had been in water since
May 10. Dull sky. yf 80-88 per cent.
T 12:6-13°8° C.
of transpiration for the ight and dark periods is given as before :—
Time. Rate. Time. Rate.
i Pee |
Light. 2.36 P.M Dark.
10.41 A.M. S 3.52 ,
10.56 ,, MD Bian hos
10.57 =, Dark. 3.56 ,, Light.
11.45 ,, 6.9 :
ivedne 1) 613» 128
11.56 ,, Light.
11 PM
2:34 172
The average rate
The Effect of Inght on the Transpiration of Leaves. 289
The average of the three light readings is 160, of the two dark ones 122
or L/D = 160/122 = 131/100.
If the L reading for 6.9-6.13 P.M. is omitted, which is fairer owing to the
fading of the light, we have L/D = 176/122 = 144/100.
Experiment 18.—May 14, 1915. P. lawrocerasus.
10.20 a.m.—Branch, bearing last year’s leaves, vaselined and fitted to
potometer.
11.2 a.mM.—Incisions made in leaves. North window, fair light. yy 71-78 per
cent. T 15-2-15-9° C.
| Time. Rate. Average. Corrected for w.
| 11.45 ae
3 A.M.
} |
ee ew 2
1152 ,, (as cneee
Die | tee :
Pe Sea by eee Ll Bee aus
12.28 ,, | Light, fairly bright.
| 4.44 ,, | 202
BAD | CASE 1966)1 | 190
451 ,, | 194 |
The average of the two L periods is 209, which is practically equal to the
transpiration in the D period. This result is not explicable, as the light
was good at 4.44. Nor was there any evidence of the wood-yvessels being
blocked, as sometimes occurs in potometer experiments.
Average: L/D = 209/208 = 100-5/100-0.
Experiment 19.—May 17,1913. P. lawrocerasus.
Fresh surfaces cut to the branch on May 15, May 16, and May 17.
9.30 A.M.—ypr between 76 and 82 per cent. T 13-7-14:4° C.
Time. Time. Time.
| ‘ |
Light. Cirea 10.45 aM. Dark, | 11.34 a.m. Light.
10.39 am. 7 ETS) 3 ~ } 2.0 P.M. ‘
iret 26 (HBO } Ue | 2.20 ,, ee
The average of the two light periods is 194. L/D = 194/105 = 185/100. ©
The following experiments were made by Miss Pertz on P. lawrocerasus
growing at the Botany School, Cambridge :—Shoots bearing five to seven
Y 2
290 Sir F. Darwin.
leaves of the current year were cut under water, and fitted to the Bausch
and Lomb (Prof. Ganong’s) potometer. The leaves were vaselined and cut
as usual, the incisions being usually four in number. The surface of the
branch was always carefully greased.
The first column gives times of observation. The second the number of
seconds occupied by the absorption of one degree of the potometer,
ie. of 0:01 cc. The third column (R = rate) gives the reciprocals of the
time readings (Column 2) multiplied by 10,000. Thus the hourly rate of
absorption (R) is expressed in units of 0:0036 c.c. The fourth column gives
the rate R corrected for yw.
Experiment 20.—June 9, 1913. P. lawrocerasus.
Seven leaves. Relative humidity (y) varying between 58 and 63.
Temperature between 16°5 and 18:3.
Time. Time in seconds. | R. | Corrected.
10.14 a.m. | Leaves greased. Cut: placed at east window.
1018 ae 96 104 ‘0 108 0
(bright day)
102650 102-0 98 0 | 98 0
10.35, 105 -0 95 °2 102 ‘0
10.52 ,, 92°53 108 -0 | 115 °0
1 ee 100-0 100-0 100-0
Lis a 107 0 93 °5 | 93 “4
TET 4 107-0 93 °5 | 93 *4
Wats} 5 Placed in dark room.
1130) 128 0 78-1 85 °4
11.42 ,, 141 °0 70 °9 17 °5
Ilays) 5. 160 °0 62°5 68 °3
12.38 P.M. 161°0 62°1 70:0
125 ae ieadl 175 ‘0 57-1 62 *4
12:20° ,, | Replaced at east window, rather dull.
12 | 135 ‘0 | 74-0 76 ‘0
1237, 126 -0 79-4 | 81-0
12.44 ,, | 110-0 90-9 | 94.°9
|
If we take the last (corrected) reading in each period we get—
Light. Dark. Light.
oA 62-4 94-9
or 150 100 152
Average: L/D = 151/100.
The rest of the experiments are given in abbreviated form, 7.e., merely the
rate R corrected for difference in ‘
The Effect of Light on the Transpiration of Leaves. 291
Experiment 21.—June 6, 1913. P. lawrocerasus.
9.30 A.M.—Cut a shoot with nine leaves of current year and fitted to
potometer after vaselining and cutting. Dull day.
ar 59-62 per cent.
At east window.
| Time. R. | Time. | Re
|
| |
| 10.84 a.m. 61-0
ee eytO.Sye 98 °5 | 11.42 a.m, ‘In dark room.
entGis 2: a 81 °3 |p 1alaS 41 °9
(lighter) || 123 PM. | 44. °8
lie Pale Abe 69-0 iy 1220 a9) 37-7
eOn & 54°3 12.38, | 29-3
Result: L/D = 185/100.
Experiment 22.—June 7, 1913. P. lawrocerasus.
10 a.M.—Young shoots, six leaves. At east window. Dull day. w62-
74 per cent.
Time. R. Time. | R.
10.15 a.m. 49°5 || | Placed in dark room.
10.25 ,, 48 ‘0 11.39 am. | 49 “8
10.51 __,, UAO | We | 48 ‘0
Pye 66:7 || 12.12 p.m | 40-1
TP 5 59 +4, 12.24 ,, 38°38
TL) 58 ‘8 |
Result : L/D = 154/100.
Experiment 23.—June 12,1913. P. lawrocerasus.
11.50 a.m@——Young shoot, seven leaves, vaselined but no incisions made.
af 52-63 per cent.
|
|
T 17:9-20°3.
Time. R. Time. R.
|
| |
June 13, 1913. | Hi eels ame Rents 107 ‘0
9.30 am. | Fresh surface tobranch || 11.13 _,, 97 °4
| eut under water: ILA 88-7
SHA op | 25 °8 Hy alheh) 87 °8
LOOn | 24°3 | 11.48 ,, 89 2
WOE ep Cut leaves, z.e. usual || 11.54 _,, 89°9
incisions made. 11.55 ,, | Replaced at east window.
MOD. py | 69:6 12.2 P.M. 85 °5
10.10 ,, 85 °5 12.12 ,, 998
10.36. ,, 109-0 12.29 ,, 969
1042) 113-6 12.38 ,, 105 ‘0
LOA Taree hile 113 6 12.47 ,, 105°0 |
10.55 ,, 117 6 12.50 ,, 108 ‘0
10.56 ,, | Placed in dark room.
|
Result :
Light 131, Dark 100, Light 120. Average: L/D = 126/100.
292 Sir F. Darwin.
Experiment 24.—June 13,1913. P. lawrocerasus.
June 13, 1913.—A shoot with nine leaves, none very young; vaselined
June 14,10.5 am. At east window. wy 63-61 percent. T 18-4-20° C.
Time. R. | Time. R.
| |
|
10.19 a.m. 23 °°9 11.32 a.m. 128
10.24 ,, 26 5 11.38) ,, 131
10.26 ,, Cut up leaves. MAe/ 6 126 |
WO sp 95 °2 11.56 ,, 129 |
10.34 ,, 126°6 12.11 P.M. 118
10.37 _,, 140 NPI] — Replaced at east window.
10.50 ,, 150 12.18 ,, 128
We 4 160 12.23 |, 137
THO, 4 154 12.30 ., 138
11.15 ,, 156 12.36 ,, 1384
ING 55 Placed in dark room. TPG) 1, 140
(ese 144
Result :—Light 132, Dark 100, Light 119, Average: L/D = 126/100. |
Experiment 25.—June 16,1913. P. lawrocerasus.
Shoot with seven leaves, none very young. Vaselined and placed at east
window.
Time. R. Time. R.
June 17,1913. | Cut fresh surface to | 10.36 a.m. 102 ‘0
branch under water 10.45 ,, 105 :0
9.85 A.M. | Placed at east window.|| 11.4 _,, 105 ‘0
| y 56-66 per cent. | DS 18 Placed in dark room.
| | 219-25 °8 Semi on 75 °3
| 9.59 ,, | 19°8 Per ealiecya ys as 76°1
1@@ 5, Cut up the leaves 11.42 ,, 78 °6
TOP 5, 67:1 las eatieG One® 76°7
10.10 ,, 95 °5 11.52- ,, Replaced at east window.
10.14 ,, 105 ‘0 12.23 P.M. 106-0
ee TODS), S 109-3 TOs, 106 ‘0
10.30 ,, 103 -0
Result: Light 188, Dark 100, Light 187. Average: L/D = 138/100.
Weighing Experiments (Laurel).
In a few experiments the transpiration was estimated by the loss
of weight of a cut branch (laurel) in a bottle of water covered with a layer
of olive oil, The branches had each six leaves, which were carefully
vaselined and cut in the usual way. The stem and all buds also vaselined
with care.
The Effect of Inght on the Transpiration of Leaves. 293
The experiments took place inthe laboratory above referred to, in which
darkness was not absolute.
The specimens weighed from 100 to 130 erm., and were only weighed to
within 5 mgrm.
Experiment 26.—May 10, 1913. P. lawrocerasus.
8.52 am —At bright north light. yw 71-88 per cent. T 11-13°6° C.
| |
| Loss. |
Time.
| Per hour. Corrected for w. |
| |
|
9.37 on. |
B AM o | 2 |
Bes } Light 0252 219
1OS2ee ae :| : OP |
ions } Dark 0:114 127
IIA oe - 5
Aan ee } Light | 07180 167
Se Dark 0-107 122
iS aaa |
Weg! | Light 0-244 217
rte ae | | (sky very bright.)
Ao } Dark | 0-113 | 129
Result, average L/D = 201/126 = 159/100.
Experiment 27.—May 22,1913. P. lawrocerasus.
Leaves vaselined, but not incised until May 23.
May 23.—Light clouds. wW 75-86 per cent. T 14:2-18:2° C.
| | Loss
Time. |
| Per hour. | Corrected for y.
| orm.
Bre: si \ Light 0-249 271
epee p } Dark 0-134 167
Bie ee } Light | 0-294 254
ae : } Dark | 0°250 208
Bee : } Light | 0-242 242 |
aie : | Dark | 0 +236 197
Average: L/D = 256/191 = 134/100.
294 Sir F. Darwin.
Experiment 28.—May 24, 1913. P. laurocerasus. fy 77-88 per cent.
T 15-1-18°5° C.
Loss
Time. SSS a ==
Per hour. Corrected for yw.
grm
ae a } Light, 0-328 290
aa \ Dark, 0-129 153
een : } Light. 0-321 273
ee 2 \ Dark, 0-195 163
ae p } Light. 0-217 217
Average : L/D = 245/158 = 155/100.
Or, omitting the last L period: L/D = 325/162, or 201/100.
Experiment 29.—May 25. P.laurocerasus. ry 76-83 per cent. T 17:8-20°5.
|
Time. Rate. Corrected for i.
ee ae
| oun } Tighe el 231 231
ee z \ Dark 155 162
sea g \ Light | 264 | 264
es : } Light 257 236
| Average: L/D = 244/162 = 151/100.
The results of the series of four weighings are :—
L/D = 159/100 ; 134/100; 201/100; 151/100. Average L/D = 161/100.
Experiments on Ivy (Hedera helix).— July, 1913.
In the following experiments by Miss Pertz the specimens were cut at
night and placed in water, and on the following morning a fresh surface was
cut under water.
The leaves and stems were then carefully vaselined and four incisions
per leaf were made.
In all cases the transpiration rate is corrected for wp.
The Effect of Light on the Transpiration of Leaves. | 295
Experiment 30.—July 1, 1915. Ivy.
10.20 a.m.—Shoot with seven leaves vaselined, no incisions made.
July 2, 9.30 amM.—In potometer at east window. Dull. During the day
day yr 60-70 per cent. T18-19°6° C.
\]
Time. | R. Average. || Time. | R. | Average.
9.38 A.M. mr | : 10.43 A.ot 17-0
9M 27-8 | GOs 71-9 |
9.43 ,, Cut up leaves with scalpel, | 114 ,, 78-2 |
4, cuts each leaf. ie alla 74.6 \ 76
ain. Tere. Teter ep ell 76°9 |
Creal 82-0 i ALT) | Replaced at east window.
LOO 96 -2 Wea ateo ot sen ta! 75°8
TON. 91-3 Ww iaeoy aenet| 82-0
10.26 ,, 1120 || 1243 Pm. | 91-0
He 0332 1240 12.46 ,, | | 91-0
dogs 2 106-0 } We 12.52 | 99-0 2
10.35, Placed in dark room. |
|
L. D. L.
Result 115 76 95 Average: L/D = 138/100.
or 151 100 125
Experiment 31.—July 4, 1915. Ivy.
11 A.M.—Shoot, 12 leaves, cut and vaselined.
July 5, 10 am—In potometer at east window. during day 63-68 per
cent. T16°7-18:4° C.
Time. | R. Average. | Time. R. | Average.
| Fie |
i
10.15 a.m. 32°0 Hmloleede jaea te 121 °0
LONG 8 207 ie) op 106-0
10.22 ,, | Cut up leaves with scalpel, 11.51 ,, 102 °0 103
4 cuts each leaf ieee Oe 104 0
10.25 _,, 108-0 | 12.0 noon | Replaced at east window.
10s © 122-0 | 12.8 p.ac. 1160 |
1040 126 ‘0 eeiokienare 123-0 |
10.52 ,, 13700 | | ees 5 125-0
a 139 0 i ie 127°0 Ci
On ae 147-0 || a 12/47 5, 1230 |
Ale eee || geen O 9 EH 131-0 |
Tet oee een eEIcodiinidarie room mE |) | rea } ie
i | |
L. D. L.
Result 147 103 133 Average: L/D = 136/100.
or 143 100 129
296
Sir F. Darwin.
Experiment 32.—July 7, 1913. Ivy.
11 a.m.—Shoot cut, 10 leaves (current year) vaselined.
July 8, 9.55 4m.—In potometer at east window. during day 54-64 per
Centan eh Lo2= toi,
| | | |
. Time. R. | Average. Time. R. | Average.
b Bs
10.5 A.M. 21°5 11.2 aM. 115-0 |
109 20 °7 cibists oy 111-0
10.11 ,, Cut up leaves, 4 cuts per 1425) 5; 118-0
leaf. IDES IE 120-0 119
LOWSS ; 85°d iH h3%) yy | 118°0
WO, 100 ‘0 ibbsby | Replaced at east window.
10.24 ,, 109 ‘0 11.46 ,, 1330
10.32 ,, 125 0 Sas 133 °0
| T@3i) 5, 127°0 | 12.2 P.M 138 °0
10.47 ,, 125 °0 | 129 12.12 ,, 140-0 |
10.49, 132-0 12.19 ,, 140°0 | 18a
10.50 _,, | Placed in dark room. 5 by ee | 188 -0 |
We D. L.
| Result 129 119 139 Average : L/D = 113/100.
or 108 100 117
Experiment 33.—July 9, 1913. Ivy.
10 a.m.—Shoot having 13 leaves of current year, cut and vaselined.
July 10.—In potometer at east window. during day 65-735 per cent.
T 16°7-19:2° C.
Time. R. | Average. Time R. | Average.
10.11 A.M. Cut up leaves, 4 cuts each | 11.51 a.m 120 | 121
leaf. 11.56 ,, 121 |
10.17 ,, 96 HME» 5 Replaced at east window.
10.26 ,, 110 12.4° PM 150
TOES) 130 i208 5 154
TOv | 147 | 12.16 ,, 157
10.51 ,, 143 12.22 ,, 152
TOR? 135 | 140 UP, or 154
10.59, 145 japan 151
TL) Placed in dark room. | 12.35 ,, 159
Taal 112 | Hh BS \ 149 \ as
11.49 ,, 123 |). 12.89 % 157
Ibi. D. L.
Result 140 121 153 Average: L/D = 121/100.
or 116 100 126
The Effect of Light on the Transpiration of Leaves. 297
Experiment 34.—July 10,1913. Ivy.
Shoot having nine leaves, six being of current year, vaselined.
July 11, 10.20 am—tIn potometer at east window. wduring day
66-69 per cent. T17-189° C. Dull morning.
| Time.
Nae Average. | Time. R. Average.
| | |
| . 10.29 a.m. | 22 °6 11.52 A.M. 71:0
10.31 ,, Cut up leaves, 4 cuts each 12.6 P.M. 75 0 | 75
| leaf. WAMSy 74:0
1034 °,, | 88-0 WW 5 Replaced at east window.
10.40 ,, j 83-0 | Kgl 232 Gia 80-0 |
10.49 ,, | 85 ‘0 | PEP 91-0 =|
ie 55 98-0 12.44 ,, 89:0 |
11.22 ,, 93-0 | a 12.59 ,, 9-0
125. 97-0 | Pepe ras ees 94:0
E26, | Placed i in dark room. alg eee 103 0 | 101 |
TESA 82-0 | NAB 98 -0 |
ior ;, 76-0 |
|
| oy
Th D. Tt
Result 95 75 101 Average: L/D = 131/100.
or 127 100 135
Experiment 35.—June 22,1913. Ivy.
The followimg experiment may be placed with the above, although
transpiration was estimated by weighing instead of with the potometer.
The method was the same as that described for laurel.
June 21, 4.30 p.wm—Branch cut under water and placed in water covered
with a layer of oil. The lower surfaces of the leaves (of the current year)
carefully vaselined, together with the stem.
June 22, 9.40 a.m.—Four or five incisions made per leaf. Placed in north
window ; the sky fairly bright. from 72 to 80 per cent. T 166—19-2° C.
| |
| ies ee mee | Rate corrected
kee
10.18 a.m. F 12.34 P.M. j ° 5
| 1120 Light | 295 137 ii Light | 361
rine) ob 137 ,, ted
| TSE Dark 187 ee } Dark 190
The average transpiration L/D = 328/189
or L/D = 174/100
The average of the L/D results for ivy, viz. 138, 136, 113, 121, 131,
174/100, is 136/100.
298 Sir F. Darwin.
Results.
§ 1. The method employed was to close the stomata by carefully rubbing
the stomatal surface with cocoa-fat or vaseline, the intercellular spaces being
afterwards put in communication with the outer air by means of incisions.
In the case of leaves not thus treated, it is well known that the closure of
the stomata in darkness greatly diminishes the evaporating surface and
vice versdé. In my method the evaporating surface is a constant.
§ 2. The following tables give the comparative effects of diffused daylight
and darkness on the transpiration of P. lawrocerasus and Hedera helix treated
as in § 1.
P. laurocerasus.
Date. | —_ Light. | Dark. | Date. | Light. Dark.
== eee = 3 | |
1911 | 1913.
AoA WL Sooo 105 100 i Misiy Air as | 101 100
ae Osta 100 100 Se d/e eaote 185 100
RRO otk 148 100 ee 134. 100
Ne nite 123 100 | Sh oe tebe 201 100
1913. 3), (AO Beieaiaes | 151 100
AMO NL ocece . drame) G)soress | 185 100
May 808 } wee LOOK all> Sista kiana | 95a. | ioe
ae eer ted wea 116 100 In cae SO ences 151 100
PACES & acts 107 100 1 pao eee 126 | 100
) 6: .c8igs3 125 100 oe Styyattne 126 100
he toes 114, | 100 pt hare secee 138 | 100
fy: fhaborenc 134 100 1911. |
p cS cero 100 | 100 INOW UE) Goosen 125 | 100
a lO ee 105 100 Viiael das Uy ele cae 114 | 100
sity lO we oe 159 100 1909. |
ais aeee 120 | 100 i Dec. Ob Ae. 119 | 100
4) | Zia 144 100 i}
| | | |
Average L/D: 131°7/100, in round numbers 132/100 or 32 per cent.
* The average of eight experiments, see p. 286.
Ivy (Hedera).
| |
Date. Light. | Dark. Date. | Light. | Dark.
|
: | z ane :
June 2251913, | lz) Mon July 7,1918' 118 100
Sulys as S| 138 | 100 x bt 121 100
* 4s 136 | 100 ay Alo} 5 | 131 100
| | |
Average L/D : 136/100 or 36 per cent.
§ 3. The tables given under § 2 show a remarkable degree of variability :
the extreme cases are: April 19, 1911, when the result was nz, and May 24,
The Effect of Inght on the Transpiration of Leaves. 299
1913, when the transpiration in light was double that in darkness. The
average ratio for transpiration in light and darkness is: ivy, 136/100;
laurel, 132/100. But between May 14 and June 16 the laurel gives an
average 150/100, and, speaking generally, it cannot be doubted that the
laurel reacts to illumination more in early summer than in spring. The
winter experiments are not sufficiently numerous to justify any comparison
with those obtained in summer.
It is at present impossible to form any conclusion as to the cause of the
increased reaction in June. I have no evidence as to whether the
increased permeability to water is a periodic effect, or connected with the age
of the leaf, or with the brightness of the summer sky, as compared with
illumination earlier in the year.
§ 4. With regard to the main fact that transpiration is increased by light
or diminished by darkness, we may either accept the view of Wiesner,* viz.,
that in lght the chloroplasts are warmed by the absorption of radiant
energy, or we may believe that light produces an increased permeability of
the plasmic membrane to water, a point of view to which the interesting
work of Lepeschkin and Troéndle+ on the increased permeability to dissolved
substances produced by illumination may possibly give some support. Or we
may combine Wiesner’s theory with those of the other writers.
It is a pleasure to express my thanks to Miss D. F. M. Pertz for the
valuable aid she has given me throughout the research.
* Wiesner, ‘Sitzb. d. k. Akad. Wiss.,’ 1877, vol. 74, p. 477.
+ Lepeschkin, ‘ Ber. d. Bot. Ges.,’ xxvi, a; Trondle, ibid. xxvii.
300
The Chenncal Interpretation of some Mendelian Factors for
Fiower-Colour.
By M. WHELDALE, Fellow of Newnham College, Cambridge, and
H. Lu. Basserr, Trinity Hall, Cambridge.
(Communicated by W. Bateson, F.R.S. Received November 21, 1913,—Read
January 22, 1914.)
(From the Laboratory of the John Innes Horticultural Institution, Merton, Surrey, and
the Balfour Laboratory, Cambridge.)
The inheritance of flower-colour in Antirrhinum majus has been worked
out by one of us* and also by Baur.t Investigation has shown that the
flower-colour of the type in Antirrhinum is due to the presence of at least
six factors and that these, in various combinations, produce a series of colour-
varieties. Full accounts of the factors have been given in the papers cited,
but for convenience of reference four are mentioned again here, 7.¢. :—
Y. A factor representing the power to form ivory pigment in the tube,
accompanied by yellow pigment in the lips.
I. A factor representing the power to form ivory pigment in the lips.
R. A factor representing the power to form red pigment in the flower.
B. A factor representing the power to convert red into magenta pigment.
The factorial constitution of the type and varieties can be expressed as
follows :-—
NOAA aia) 119) 15119)) Mes cain bseahe Yellow.
YY(y)U1@)rrB(b)B(b) ......... Ivory.
MAU GPWEW EXOD cocbocasensocs. Bronze.
VY HG RR@ bes. oss Red.
VY G@)wRR@) DB (o) Ree cer. Crimson.
YONG) WG @) BIB (b) pees Magenta.
yyl(i)I(i)R(x)R(z)B(b)B(b) ... White.
In 1909 a series of researches was commenced with a view to the inter-
pretation of the above factors in terms of chemical substances, or possibly
* Wheldale, M., “The Inheritance of Flower-colour in Anturrhinwm majus,” ‘ Roy. Soc.
Proe.,’ 1907, B, vol. 79, p. 288; “Further Observations on the Inheritance of Flower-
colour in Antirrhinum majus,” ‘Rep. Evol. Com. Roy. Soc.,’ V, 1909, p. 1.
+ Baur, E., “Einige Ergebnisse der experimentellen Vererbungslehre,” ‘ Beihefte zur
Med. Klinik,’ Berlin, 1908, Heft 10, p. 265; ‘“ Vererbungs- und Bastardierungsversuche
mit Antirhinum,” ‘Zs. indukt. Abstammungslehre,’ Berlin, 1910, vol. 3, p. 34.
Mendelian Factors for Flower-Colour. 301
enzymes. Since some of the pigments involved (red, magenta) belong to the
group of so-called anthocyanins, 7.c. soluble red, purple and blue pigments of
plants, general investigations were at first made by one of us* on anthocyanin
pigments. As a result of qualitative reactions, in conjunction with evidence
from cross-breeding, it was suggested that anthocyanins, as a group, are
oxidised products of the natural yellow colouring matters, the flavones and
xanthones. At the same time it was pointed out that a number of the latter
substances had been isolated by Perkin and others} from various plants and
several had been shown to be widely distributed. The existence of many
flavones and flavone derivatives was mentioned and attention was drawn to
the fact that, as a group, they have similar properties but differ among
themselves in the number and position of their hydroxyl groups and in other
points. It was further suggested that the oxidised products (anthocyanins)
might, in a similar way, form a group of closely related substances, differing
individually according to the flavone from which each had been derived.
In view of evidence collected from various sources, it was again suggested
by one of us,{ that since the flavones are known to be present in many cases
as glucosides in the plant, the reactions involved in the formation of
anthocyanin might be stated in very general terms as follows :—
Glucoside + water = flavone + sugar,
x (flavone) + oxygen — anthocyanin,
and also that, in addition to oxidation, there might be condensation of the
flavone molecules. It was likewise stated that the first reaction might be
controlled by a glucoside-splitting enzyme and the second, if due to
oxidation, by an oxydase.
Subsequent work has strengthened the view that anthocyanins are, in all
probability, derivatives of the flavones, though we ourselves have no further
evidence as to the actual nature of the reactions involved in their formation.
Since we find little reliance can be placed on results given by crude water
or alcoholic extracts from flowers, in all later investigations an attempt has
been made to deal with the isolated and purified pigments. In a paper by
one of us,§ the methods of preparation and purification of the crude pigment
* Wheldale, M., “The Colours and Pigments of Flowers with special Reference to
Genetics,” ‘Roy. Soc. Proc.,’ 1909, B, vol. 81, p. 44; “On the Nature of Anthocyanin,”
“Phil. Soc. Proc.,’ Cambridge, 1909, vol. 15, p. 137.
+ Perkin, A. G., various papers in ‘Chem. Soc. Trans,’ from 1895 to 1904.
{ Wheldale, M., “On the Formation of Anthocyanin,” ‘Journ. Genetics,’ 1911, vol. 1,
p. 131.
§ Wheldale, M., “The Flower Pigments of Antirriinum majus. I.—Method of Prepara-
tion,” ‘ Biochem. Journ.,’ 1913, vol. 7, p. 87.
302 Miss M. Wheldale and Mr. H. Ll. Bassett.
have been described. In a more recent paper by both authors,* an account
has been given of the identification of the ivory pigment of Antirrhinwm with
apigenin, a flavone of known constitution, isolated by Perkin} from apiin, a
glucoside occurring in the parsley, Apiwm petroselinum. Apigenin is a very
pale yellow crystalline substance, readily soluble in hot alcohol, slightly so
in ether and almost insoluble in water. Melting point, 347° C. In the
Antirrhinun plant, apigenin undoubtedly exists as a glucoside, in which state
it is more soluble than after hydrolysis.
Attention has been given subsequently to the yellow pigment and the
results are included in the present paper. The crude pigment prepared from
yellow flowers was extracted with ether by methods described in previous
papers. The ether extract contains apigenin from the tube and inner tissues
of the corolla, and yellow pigment from the epidermis of the lips, including
the patch on the palate. It was at first thought that the yellow pigments in
the epidermis of the lips and in the patch on the palate might be identical.
After removing the bulk of the apigenin from the ether extract by crystallisa-
tion from alcohol, the remaining yellow pigment, which is very soluble in
alcohol, gave, on fractional crystallisation from dilute alcohol, products of
which the melting points varied from about 250° to 338° C.
The wide range of the melting points, combined with certain qualitative
reactions of these extracts, led to the conclusion that the palate contained
the lip pigment mixed with other pigments, or even other pigments without
the lip pigment. Since, however, the patch on the palate is common to all
varieties (except white), the factorial difference between ivory and yellow is
only concerned with the yellow lip pigment. Hence, in order to simplify the
problem, the pigments of the palate have been disregarded for the time
being, and investigations have been limited to crude material (unfortunately
prepared only in small quantity) from the upper lips of the yellow
variety. In this product, it seemed more likely that there would only be
two pigments present to any extent.
Even the more simple mixture presented very great difficulties in the
separation of yellow from ivory, both pigments having almost the same
solubilities in all solvents used. Such separation as was possible by means
of different solubilities gave products which indicated by their melting
points, 300-328° C., the presence of luteolin, this substance being the only
* Wheldale, M., and Bassett, H. Ll., “The Flower Pigments of Antirrhinum majus.
II.—The Pale Yellow or Ivory Pigment,” ‘ Biochem. Journ.,’ 1913, vol. 7, p. 441.
+ Perkin, A. G., “Apiin and Apigenin,” ‘Chem. Soc. Journ., Trans.,’ 1897, vol. 71,
p- 805 ; 1900, vol. 77, p. 416.
Mendelian Factors for Flower-Colour. 303
known flavone melting above 300° C. and having at the same time the
solubilities and properties of the yellow pigment.
Proceeding on the assumption that the yellow pigment might be luteolin,
a fairly satisfactory separation was brought about by hydrobromic acid,
which, according to Perkin,* forms, in glacial acetic acid, a compound with
luteolin but not with apigenin. The luteolin hydrobromide remains in
solution unless excess of hydrobromic acid is added, when it separates out in
_ochre-coloured crystals which are decomposed by water into luteolin and
hydrobromic acid. The method of procedure in our case was as follows: The
ether extract containing the mixed pigments was ground into a thin paste
with glacial acetic acid, heated to boiling, and hydrobromie acid added, but
not in excess. On cooling, the bulk of the apigenin separated out, while the
yellow pigment remained in solution. The apigenin was filtered off, and on
addition of much water to the filtrate the yellow pigment separated out and
was also filtered off. A repetition of this process ensures greater purity of
the yellow. After drying, the yellow was further purified by extraction with
ether.
The pigment prepared in this way, except for its melting point, which
varied from 310° to 328° C., resembled luteolin in properties. According to
Perkin,} luteolin is a bright yellow crystalline substance, readily soluble in
alcohol, fairly soluble in ether, and very slightly soluble in water, even when
hot. With ferric chloride solution luteolin gives at first a green, later a red-
brown, coloration. The melting point of luteolin was for many years given
as “above 320° C.” More recently Perkin has obtained luteolin by two
different methods of purification, giving, in one case, a product melting at
321-329- C., in the other, at 323—326° C.
Luteolin oceurs in Genista tinctoriat and in leaves of Digitalis§ and also,
together with small quantities of apigenin, in Reseda luteola.||
The structural formule of luteolin and apigenin are as follows :—
* Perkin, A. G., “ Luteolin.—Part I,” ‘Chem. Soc. Journ., Trans.,’ 1896, vol. 69, p. 206.
+ Perkin, A. G., “ Luteolin.—Part I,” ‘Chem. Soc. Journ., Trans.,’ 1896, vol. 69, p. 206 ;
“ Tuteolin.—Part II,” ‘Chem. Soc. Journ., Trans.,’ 1896, vol. 69, p. 799; Perkin, A. G.,
and Horsfall, L. H., “Luteolin.—Part III,” ‘Chem. Soc. Journ., Trans.,’ 1903, vol. 77,
p. 1314.
t Perkin, A. G., and Newbury, F. G. “The Colouring Matters contained in Dyer’s
Broom (Genista tinctoria) and Heather (Calluna vulgaris),” ‘Chem. Soc. Journ., Trans.,’
1899, vol. 75, p. 830.
§ Fleischer, F., “ Digitoflavon, ein neuer Kérper aus der Digitalis purpurea,” ‘ Ber. D.
Chem. Ges.,’ 1899, vol. 32, p. 1184; Killiani, H., u. Mayer, O., “ Ueber die Identitit von
Digitoflavon und Luteolin,” ‘ Ber. D. Chem. Ges.,’ 1901, vol. 34, p. 3577.
|| Perkin, loc. cit.
VOL. LXXXVII.—B. Z
304. Miss M. Wheldale and Mr..H. Ll. Bassett.
O pals 08
Co =e ju
SAN
HOP COe- ean %
Apigenin. Luteolin.
As pointed out by Smiles,* the more intensely coloured flavones contain
two hydroxyl groups in the ortho position with respect to one another,
whereas the arrangement in apigenin is not so productive of colour.
The close connection between the structure of the two substances, and the
fact of their occurrence together in Reseda luteola, also favour the assumption
that the yellow Antirrhinum pigment is luteolin. The presence of luteolin
in the allied genus Dzgitalts is also of interest.
In order to corroborate this suggestion, attempts were made to form both
the acetyl and benzoyl derivatives of the yellow pigment. The attempts
failed, owing partly to the small amount of pigment available, and partly to
the following difficulties. In the case of the acetyl derivative, the method of
dissolving the pigment in caustic soda or pyridine and adding acetyl chloride
apparently failed to acetylate the pigment completely. The method
employed by Perkin and others of boiling the pigment with acetic anhydride
and anhydrous sodium acetate was not found satisfactory when dealing with
such small amounts of substance, since there were produced simultaneously
brown decomposition products, from which it was impossible to isolate the
derivative. In attempts to benzoylate the yellow pigment by the Schotten-
Baumann method, the same difficulties arose, together with a further one,
namely, the fact that the melting point, 201° C., of the benzoyl derivative of
luteolin is only about 10° lower than that, 210-212° C., of the benzoyl
derivative of apigenin; hence the possibility that the small amount of
product formed might be impure apigenin derivative produced from apigenin
retained in the luteolin used. It has been shown by Perkinf that in the
Schotten-Baumann method, under certain conditions, a tribenzoyl, instead of
a tetrabenzoyl, derivative may be formed.
Finally, attempts were made to form the benzol sulphonyl derivative
described by Fleischer{ as obtained from digitoflavone, the latter substance
being extracted from Digitalis leaves and subsequently shown to be identical
with luteolin. Fleischer’s benzol sulphonyl derivative was obtained by
* Smiles, S., ‘The Relations between Chemical Constitution and some Physical
Properties, London, 1910.
+ Perkin, A. G., “Notes on Luteolin and Apigenin,” ocueut Soc. Journ., Trans.,’ 1902,
vol. 81, p. 1174.
Bit rleiechen F., loc. cit.
Mendelian Factors for Flower-Colour. 305
treating the digitoflavone, in caustic soda solution, with benzol sulphochloride
and erystallising the product from a mixture of chloroform and ether.
Melting point, 189° C.
By treating a specimen of yellow pigment, purified by means of the
hydrobromide method and subsequent extraction with ether, in a similar
way with benzol sulphochloride, an almost white product was obtained,
which crystallised from a mixture of chloroform and ether and melted at
188-190° ©,
By hydrolysing a small quantity of the benzol sulphonyl derivative with
alcoholic soda for three hours, a sample of luteolin was obtained, melting at
324° C.
There is no doubt that the yellow Antirrhinum pigment is luteolin. The
factorial difference between the yellow and ivory varieties can, therefore,
be expressed, as follows :—The ivory variety has the power to form apigenin
throughout the tissues of the flower, whereas the yellow variety has the
power to form luteolin, either in addition to, or more probably instead of,
apigenin, in the upper epidermis of the lips. It appears most likely that
the yellow variety has lost the power to form apigenin in the epidermis
and produces luteolin instead, though there does not seem to be any
particular reason why the power to form apigenin, instead of luteolin, should
be a dominant character.* The different flavones synthesised in either case
may be regarded rather as an expression of a fundamental difference in
structure of the living molecule in the two varieties, affecting, perhaps, the
production of different hydroxybenzoic acids, from which the flavones may
be synthesised. Little can be gained at present by postulating the existence
of a special organic catalyst or enzyme, representing the “1” factor, and
concerned with the removal or addition of an hydroxyl group.
From the white variety no flavones could be extracted, and this is in
accordance with Mendelian evidence. We must suppose, therefore, that
either the substances from which the flavones are synthesised are absent, or
the power of synthesis fails.
As regards the yellow patch on the palate, it appears likely that other
flavones, having lower melting points and slightly deeper colour than luteolin,
are present in this region.
It seems highly probable that the anthocyanin pigments are derived from
the flavones by oxidation, or condensation, or both, though only accurate
analyses of the pure pigments can ultimately decide this question. With
regard to the suggestion made by one of us as to the mode of formation of
* There are probably very small quantities of other flayones in the lips of both yellow
and ivory, but these do not affect the mass colour of the flowers. .
Yh Pe
306 Miss M. Wheldale and Mr. H. Ll. Bassett.
anthocyanin from the flavone, 1.¢., that the hydroxyls of the flavones may
be protected by sugar, so to speak, and only after hydrolysis can changes
take place at these points, there is no very definite evidence as to the
number of sugar molecules attached to flavones in the plant. Careful
isolation and analysis would be necessary to ascertain the actual condition
in the living plant, owing to the great ease with which hydrolysis takes
place after death.
Red and magenta anthocyanin have been obtained by us from Antirrhinum
in a fairly pure state, and certain derivatives have been made. The fact
that these, as well as the pigments, are practically amorphous indicates that
they probably have very high molecular weights. The lack of melting
points in the pigments supports this view.
In a recent paper Keeble, Armstrong, and Jones* bring forward an
hypothesis to explain the loss of colour when coloured petals are treated
with strong alcohol, and the subsequent restoration of colour when they
are treated with water.
The phenomena recorded are as follows :—When coloured (anthocyanin)
petals of Stocks (M/atthiola) are placed in strong alcohol, some pigment passes
into solution in the alcohol, which at first is coloured but fairly rapidly
becomes colourless. The petals also become colourless though more slowly.
When the colourless petals are taken out and placed in water the colour
returns; in hot water the recovery is more rapid. When the extract is
filtered from the petals and evaporated to dryness on a water-bath the colour
returns to the residue. In addition we have noted that colour returns to
the alcoholic filtrate on dilution with water, and this also happens even
after evaporation to dryness and taking up again with alcohol.
The above phenomena are exhibited by most pigments of the anthocyanin
class, and have been noted by various authors working on anthocyanin,
among whom may be mentioned Hansen,t Molisch,t and Grafe.§
The hypothesis brought forward by Keeble, Armstrong, and Jones to
explain these phenomena is the following :—The petals contain an oxydase
and a reducing agent, which is probably not an enzyme. The oxydase is
responsible for the production of anthocyanin from the chromogen, and the
* Keeble, F., Armstrong, E. F., and Jones, W. N., “The Formation of the Anthocyan
Pigments of Plants. Part IV.—The Chromogens,” ‘ Roy. Soc. Proc.,’ 1913, B, vol. 86,
p. 308.
+ Hansen, A., ‘ Die Farbstoffe der Bliithen und Friichte, Wiirzburg, 1884.
{ Molisch, H. J., “ Ueber amorphes und kristallisiertes Anthokyan,” ‘ Bot. Zeit.,’
Leipzig, 1905, vol. 63, p. 145.
§ Grafe, V., “Studien tiber das Anthokyan.—Mittheilung 3,” ‘Sitzb. Ak. Wiss. Wien,’
1911, vol. 120 (1), p. 765.
Mendelian Factors for Flower-Colour, 307
reducing agent reverses the reaction. With a decrease in amount of
water in the cell the reducing agent becomes active and the oxydase inert,
but with an increase in amount of water the oxydase becomes active and its
effect is greater than that of the reducing agent. Hence, when petals are
treated with strong alcohol the oxydases can no longer function, and the
reducing agent is then able to reduce the anthocyanin to a colourless leuco-
compound. On addition of water the oxydase again becomes active and
re-oxidises the leuco-compound.
Such is the hypothesis, but we are not clear as to the explanation offered
by the authors for the reappearance of colour in the alcoholic solution apart
from the petals. Two alternatives offer themselves. First, that both oxydase
and reducing agent are extracted by 95-99-per-cent. alcohol and are present
in the aleoholic extract and that neither is affected by heating to 100° C.* (in
spite of the fact that extraction by absolute alcohol and resistance to heat is
not characteristic of oxydases), and that, although the authors quote
experiments to prove that the oxydase can oxidise to some extent in
95-per-cent. alcohol, the reducing agent is more powerful in this medium.
Or, that the reducing agent alone is extracted by alcohol and its influence
is removed by evaporating the alcohol or by diluting, when re-oxidation occurs
merely on exposure to air. If the latter be the case, the presence of the
oxydase is superfluous to the recovery of colour in the petals themselves.
We must also conclude that the reducing agent is very widely distributed, is
unaffected by temperature of 100° C., and can only act in presence of alcohol.
To us the reduction and oxidation hypothesis appears directly opposed to
essential experimental facts, although the original production of anthocyanins
in the plant is, in all probability, either partly or wholly due to the action of
an oxydase on a chromogen, most likely a flavone or xanthone,
In our experiments, various coloured petals of Stocks were used, and these
were the flowers also used by Keeble, Armstrong, and Jones.
Experimentally we found that the same results are given both by the
decolorised petals and by the alcoholic solution.
We find that if a little acid is added to absolute alcohol containing
decolorised petals, the usual red colour reaction of acid with an anthocyanin
is obtained both in the solution and in the petals. Moreover the same result
is obtained equally well when dry hydrochloric acid gas or dry hydriodic
acid gas is passed through the alcohol. Also, contrary to the observations of
Keeble, Armstrong, and Jones, we find that prussic acid gas acts quite as
* In a later paper (Jones, W. N., “The Formation of Anthocyan Pigments, Part V.—
The Chromogens of White Flowers,” ‘ Roy. Soc. Proc.,’ 1918, B, vol. 86, p. 318) the author —
definitely states that oxydase is destroyed by boiling 50-per-cent. alcohol.
308 Miss M. Wheldale and Mr. H. Ll. Bassett.
/
well as any other acid, which would not be the case if an enzyme were
responsible for the restoration of colour. :
In any of these cases when the anthocyanin restored by acid is made
alkaline, the greenish colour reaction of anthocyanin is obtained, showing
that the restored colour is actually due to anthocyanin. The greenish
reaction is also produced directly when a drop of a solution of caustic soda
in absolute alcohol is added to the alcohol containing the decolorised petals.
If water is boiled to expel oxygen and carbon dioxide, and, while still hot,
a stream of hydrogen is bubbled through it, this water, while the hydrogen
is still passing, restores the colour to decolorised petals. In this case the
medium is neutral.
It is not conceivable that oxidation can take place in all these experiments,
particularly in that with dry hydriodie acid gas. Clearly also water is not
necessary for the change, and another explanation for the restoration of
colour must be sought.
Further, if reduction is the cause of decolorisation, the conditions in some
of these experiments are exactly those most suited for the continued stable
existence of the leuco-compounds, so that it would seem that this explanation
must also be abandoned.
There may be a reducing agent present in the petals, but its reducing
power cannot be responsible for the loss of colour in alcohol.
In support of their theory that reduction is the cause, Keeble, Armstrong,
and Jones, in a later paper,* quote the fact that an extract from the petals is
reduced to a colourless state by treatment with zinc dust and acid, and that
the colour is restored by exposure to air. We would note in passing that
this does not seem to be simply a reducing action, as we find that the
restored colour is much fainter with acetic than with sulphuric acid. This
observation has been made previously by Kastle,t who also does not consider
it simply a reducing action. Untreated anthocyanin gives exactly the same
colour with acetic as with sulphuric acid.
A point we wish to emphasize, however, is that we find the slightly acid
solution to be easily decolorised by warming with a little hydrogen peroxide
and colloidal platinum. The colourless oxidation product so formed is
unstable, and the colour is restored if the solution is made more strongly
acid.
* Keeble, F., Armstrong, E. F., and Jones, W. N., “The Formation of Anthocyan
Pigments in Plants.—Part VI,” ‘ Roy. Soc. Proc.,’ 1913, B, vol. 87, p. 113.
+ Kastle, J. H., “‘A Method for the Determination of the Affinities of Acids Colori-
metrically by Means of certain Vegetable Colouring Matters,” ‘Amer. Chem. Journ.,’
1905, vol. 33, p. 46.
Mendelian Factors for Flower-Colour. 309
Since anthocyanin can thus be decolorised by oxidation as well as by
reduction, in each case giving a product in which the colour is easily restored,
there is as much reason, on the evidence of these experiments, to postulate
one process as the other for the cause of decolorisation by treatment with
alcohol. As a matter of fact, the conditions in both experiments are so
different from those obtaining when petals are treated with alcohol, that
probably neither experiment has any real bearing on the question at all.
That an alternative to the reduction and oxidation hypothesis can be
offered, is shown by the parallel series of changes produced by using phenol-
phthalein solution, made red by ammonia, as a pigment. This red solution is
decolorised by alcohol and the colour restored by diluting largely with water
or by addition of a drop of allxali.. On evaporating the decolorised alcoholic
solution to dryness, a red residue is obtained. As it happens, phenol-
phthaleim is colourless with acids, while anthocyanin gives colour reactions
with both acid and alkali. Apart from this accidental difference, the two
cases are strikingly similar.
Without wishing to insist on the parallel too rigidly, it would seem that
the two series of phenomena might well have similar explanations, The
present authors tentatively offer two alternative suggestions without
attempting to decide between them.
It may be that strong alcohol dehydrates the anthocyanin, giving a colour-
less compound, and that colour is restored by subsequent addition of two
radicals, either H and OH, or some other pair, such as H and I, Such an
effect might perhaps be accounted for by the production in anthocyanin of a
lactone grouping. A somewhat similar explanation has been advanced to
account for the phenolphthalein changes.*
Or, the loss of colour when the petals are treated with aleohol may be due
to combination of the anthocyanin with alcohol to an unstable colourless
compound, which is easily decomposed by various reagents. A similar
explanation has been advanced by Hantzsch to account for the differently
coloured solutions given by certain substances in different solvents.
A few minor points in connection with the above work may be considered.
First, Keeble, Armstrong, and Jones state that the restoration of colour to
petals is accelerated by a drop or two of hydrogen peroxide either in acid or
alkaline medium, and, further, that the reappearance of colour is not due to
the acidity or alkalinity of the medium, because the original colour, purple,
red or pink in differently colored petals, is first restored, and the acid or
alkaline anthocyanin colour only appears later.
* Meyer, R., u. Spengler, O., “ Zur Constitution der Phtaleinsalze,” ‘ Ber. D, Chem.
Ges.,’ 1905, vol. 38, p. 1318.
310 Mendelian Factors for Flower-Colour.
Since we find that the return of colour in water is always accelerated by
acid or alkali, we suggest that the acceleration by hydrogen peroxide is
merely a function of the amount of acidity or alkalinity of the medium in
which the hydrogen peroxide is dissolved. Moreover, although the exceed-
ingly small amount of acid or alkali which at first diffuses into the petal from
the very dilute solution may be sufficient to accelerate the actual return of
colour, it is not sufficient to give the usual acid or alkaline coloration with the
anthocyanin present. Further addition of the hydrogen peroxide solution
would, and in fact does, bring about this result. In support of this, we
observe that the extract, which at once comes into contact with the full
amount of acid or alkali, immediately gives the acid or alkali colour, and not
the original purple, pink, ete., of the petals.
To confirm this suggestion we carefully neutralised some laboratory
hydrogen peroxide, which is, of course. always decidedly acid, and found that
this neutral reagent actually retarded the recovery of colour as compared
with control experiments on decolorised petals in cold, hot, or very faintly
acidified water.
This result is not surprising in view of the decolorisation of petals by
hydrogen peroxide and colloidal platinum, already described in this paper,
and, we think, clearly demonstrates that the oxidising properties of hydrogen
peroxide have nothing to do with the recovery of colour by the use of this
reagent when it has not been neutralised.
Secondly, the same authors state that the purple coloration of a petal can
be restored by re-oxidation in an acid medium. For this purpose purple
petals of Stocks are incubated with 99-per-cent. alcohol with: just enough
citric acid to render the alcohol acid. The petals become almost decolorised,
but retain a faint pink colour. When transferred to distilled water the
pigment is reproduced in considerable quantity, at first red and then purple.
We should explain the phenomenon as follows: The purple pigment is
rendered colourless by the alcohol, but, owing to the presence of a small
quantity of citric acid (which is very slightly dissociated in alcohol), the
colour does not entirely disappear, and the solution remains pink. Trans-
ference to water restores the colour, which is at first red, owing to the
increased ionisation of the citric acid by the water that soaks into the petal.
After a time the acid diffuses away into the surrounding water, leaving the
liquid in the petals practically neutral, when the pigment becomes purple.
Finally Keeble, Armstrong, and Jones note that when the colour is
restored to petals by immersion in water, and the colour is allowed to diffuse
out of them, coloration is again restored by transferring them to hot water,
and this process may be repeated two or three times.
On the Heat Production Associated with Muscular Work. 311
They hold that the successive restorations of colour are due to fresh
supplies of chromogen being produced by the plant under the influence of the
hot water, and that each fresh amount is then oxidised to anthocyanin.
We suggest that these phenomena are explained by the fact that though
a certain amount of pigment diffuses out into the water, a large proportion
of that which was originally present is retained by the coagulated proteins of
the petals, of course in the colourless state. It is the successive liberation
of fractions of this retained pigment that accounts for the fresh production
of colour in hot water, and not a new formation of chromogen.
On the Heat Production Associated with Muscular Work.*
By R. T. Giazeprooxk, M.A., F.R.S., and D. W. Dy, B.Sc.
(Received December 1, 1913,—Read January 22, 1914.)
On reading Prof. Macdonald’s paper it appeared that it might be interesting
to see if his results connecting the heat production and muscular work could
be expressed graphically or by means of some simple formula. The tables
in his paper give the heat production in calories per hour of a number of
individuals when doing a carefully measured amount of mechanical work on
a kind of treadmill or cycle. This amount of work is kept constant for each
group of observations in the paper. Table I gives his average results.
Table L.
| Heat production.
Mechanical |
work. :
From observation. From formula. |
Group A ...... 13 182 179
g 5 ee paaeey, 19 199 202
Ore 43 297 296 |
i 0 eee 56 346 | 347
On plotting these as is done in fig. 1, it is clear that the points lie very
approximately on a straight line, and it is easily seen that the equation to
this line may be written
WwW
0°256 ° @)
* A Note on Prof. J. S. Macdonald’s paper, supra, p. 96.
312 Messrs. R. T. Glazebrook and D. W. Dye.
oO 100 200 300 400
H CALORIES PER HOUR
Fic. 1.—Relation between work W and mean heat produced 18h.
(@) 100 200 300 400
H CALORIES PER HOUR.
Fic. 2.—Relation between H and W for persons of various weights M.
On the Heat Production Associated with Muscular Work. 313
or, more generally, H = Hy + (2)
where H is the heat produced, W the work done, and Hp, A constants which
have on the average in Prof. Macdonald’s experiments the values 128 and
4256. Ho is clearly the heat produced when the mechanical work done is
zero, and arises from the motion of the limbs and the processes occurring in
the body.
The fourth column of the table gives the results calculated from the formula.
But this is only an average result. It was clear from Prof. Macdonald’s
figures that the relation depended on the person doing the work, and we
proceeded to plot the corresponding curves for the various individuals. These
-are shown in fig. 2; and though of course the number of observations is not
Table IJ.—Tabulation of Experimental Results (separated out in relation to
the particular weights).
| Measurements from curves
| fig. 2.
| . Weight, M. Work, W. | Heat produced, H.
Ho. A.
eee
| kgrm. | Cals. per hour. Cals. per hour.
| 19 177
| 43-7 43 279 84 0-213
H 56 346
13 160 |
SAS 19 193 5
54°6 43 280 107 0-244
| | 56 335 |
| 13 169
eee (26) (212) .ox
55 °7 (345) (244) 114 0-250
43 ) 285
Par 13 181 ee
58 °8 { 43 298 } 130 0 °255
19 212
605 43 317 142 0-258
56 347
(| 13 186 }
19 216 |
619 «= | (34°5) (265) b= 138 0-266
| | 43 306 | }
| L 56 348 WE
| 13 209
66-7 43 324 161 0:280
56 352
The figures in parenthesis are from a paper in ‘ Brit. Assoc. Rep.,’ 1912, p. 286.
814 Messrs. R. T. Glazebrook and D. W. Dye.
very large in each case for a given individual, the relation between heat and
work is satisfied by a linear equation and can be expressed by the above
formula, with the difference, however, that Ho and » depend on the individual
and are not the same for all the persons tested. Table II gives the results
and includes figures taken from an earlier paper in the B. A. Report for 1912.
The quantity \ measures the slope of the curve. .
The next step was to investigate the relation, if any, between the quantities
Hy and X and the weight of the man denoted by M and measured in kilo-
grammes. On plotting the values of > against the mass in kilogrammes as
is done in fig. 3, we found that the points again lay very well on a straight
line and that the equation to this line was given by
A = 0:08 +0-003 M. (3)
Fic. 3.—Relation between mass M and A (slope of lines in fig. 2).
This quantity measures the ratio of the work done to (H — Hp), the heat:
employed in doing this mechanical work, and for a man of 50 kerm.
weight has the value 0°23 or nearly one-fourth ; the efficiency of such a man
is about 25 per cent.
On plotting the values of Hy against M as in fig. 4 we again found that a
simple linear relation given by
Hy = —1384+45M (4),
On the Heat Production Associated with Muscular Work. 315
satisfactorily held for all the points but one. Thus the heat a man generates
by moving his limbs in a regular manner without doing external work is
equal to the difference between 4°5 times his weight and a constant.
The one exception to the law was in the case of a boy weighing 43°7 kgrm.
who had no experience of cycling and whose earlier experiments were omitted
in consequence by Prof. Macdonald.
oO H 100 fi0 120 130 0 150 160 170
o
Fic. 4,—Relation between mass M and H, (pedals turning ; no load).
If we now sum up the results, putting the values of Hy and X from (3)
and (4) into our formula (2), we find
WwW
H = —138+45M+————___, d
+ <0" + 90840003M 2)
The curves obtained from this formula for different values ‘of W are given
in fig. 5, and the experimental results are there plotted.
816 On the Heat Production Associated with Muscular Work.
500,
|
Ran \ | =| a eats I et | >
iG) hapae ion i ae
i
300 —— ; = 2 os
o) % B
| A
weal 4 |
LY E
200-——— L |
x yh
O)
®
H die
CALS.
PER VA CURVE W
< } Fae
100 é A By ts
ae ea 7 C43
: D 56
E (0)
ee
O 1 30 40 50 60 TO 80
0) 20
M_ KILOGRAMS.
Fie. 5.—Relation between H and M calculated from equation (5).
The same results are tabulated in Table III. The differences between the
results given by the formula and those found from observation are, with
the exception of the boy of weight 43°7 kgrm., in no case large, and it
would appear that the relation
= W
H = a+0M+ ap (6)
where a, 0, a, @ are constants having for Prof. Macdonald’s experiments the
values given in formula (5), expresses, within the limits of experimental
error, the relation between the work done, the heat produced, and the weight
On the Fossil Floras of the Wyre Forest. 317
ofa man. It is clear of course that the equation cannot be pressed too far ;
as to the value of the result found, we do not feel ourselves competent to
judge. The work may, however, be of interest as an example of the analysis
of somewhat complex experimental results by simple graphical methods.
Table IIJ.—Calculated Values of H by Equation (5) for the various Constant Rates of
Work, W, used in the Experiments, and the corresponding Observed Values of same.
| Mass. W =0. IWa—liss Wi — 19! W = 48. W = 56. |
kerm, Cale. Hy. | Obs. H. | Cale. H.| Obs. H. | Cale. H.| Obs. H. | Cale. H.| Obs. H.| Cale. H. | Obs. H.
0 —138 24 99 398 562
20°0 — 48 45 88 259 352
43 °7 59 120 149 177 263 279 319 346
54°6 108 161 160 186 1938 284 280 338 335
| 55 Ti 113 166 169 190 287 285 340
| 58°8 127 178 181 201 295 298 346
| 60°5 134 184. 206 212 299 317 348 347
61°9 141 190 186 212 216 303 306 | 3852 348
l 66ic7 162 208 209 230 315 324 362 352
80°0 222 263 281 356 397
On the Fossil Flovas of the Wyre Forest, with Special Reference to
the Geology of the Coalfield and its Relationships to the
Neighbouring Coal Measure Areas.
By E. A. NEweEt Arser, M.A., Se.D., F.G.S., F.LS., Trinity College, Cambridge.
(Communicated by Prof. T. McKenny Hughes, F.R.S. Received May 20,—
Read June 5, 1913.)
(Abstract.)
In the absence of any detailed knowledge of the geology of the Wyre
Forest Coalfield, the area may be temporarily sub-divided into four regions.
Fossil floras are described from three of these: from the horizon of the Sweet
Coals in the Highley area in the north, from the unproductive beds of the Dowles
Valley in the centre, and from the horizon of the Sulphur Coals of the Southern
or Mamble area. On the evidence of the plants the Sweet Coal Series is
shown to belong to the Middle. Coal Measures, while the Sulphur Coal Series,
overlying the Sweet Coals unconformably, belongs to a higher horizon, the
Transition Coal Measures. The Dowles Valley unproductive measures
are shown to be Middle Coal Measures, which are there over 1000 feet in
318 On the Fossil Floras of the Wyre Forest.
thickness. The Middle Coal Measure flora of the Wyre Forest includes
55 species, of which three are new, two of them being new species of
Sigularia, and one,a new type of seed-impression. Four other plants are
new records for Britain. The Transition Coal Measure flora is smaller, but
includes 20 species, of which one is a new British record.
It is shown that both the Middle and Transition Coal Measures of the
Wyre Forest Coalfield consist of red-grey measures with Espley rocks. In
the Transition Coal Measures Spirorbis-limestones also occur. The distribution
of these rocks is considered in detail.
The Wyre Forest is discussed in relation to the other coalfields of the
Welsh Borderland. The lower or productive measures of Coalbrookdale, and
also the coals of the Titterstone Clee Hill are shown, on the plant evidence,
to be Middle Coal Measures. A species of Cordaicladus new to Britain is
described from the latter coalfield. It is pointed out that the Coalbrookdale-
Wyre Forest field really consists of four distinct coalfields, in part super-
imposed on one another. Two of these, the Lower Series of Coalbrookdale
and the Sweet Coal Series in the Wyre Forest, are of Middle Coal Measure
age. These are quite separate areas, and are in part overlain unconformably
by two other coalfields of Transition Coal Measures, one connecting Coal-
brookdale and the Wyre Forest, and the other confined to the southern part
of the Wyre Forest, overlying Old Red Sandstone.
It is contended that the coalfields of Shrewsbury, Le Botwood, Coalbrook-
dale, Wyre Forest, Titterstone Clee Hill and probably Newent form a related
series, which, with the exception of the Lower Series of Coalbrookdale, is
quite distinct lithologically from the Midland and Southern Pennine coal-
fields. If this is the case, it is pointed out that the theories of the originally
continuous sheet of measures, and of subsequent excessive denudation of the
Welsh Borderland, are inaccurate hypotheses, which should be abandoned.
Intermittent Vision.
By A. Mattock, F-.R.S.
(Received November 11,—Read December 11, 1913.)
[This paper is published in ‘ Proceedings, Series A, vol. 89, No. 612.] .
319
The Deternunation of the Minimal Lethal Dose of various Toxic
Substances and its Relationship to the Body Weight um Warm-
Blooded Animals, together with Considerations bearing on
the Dosage of Drugs.
By Grorces Dreyer, M.D., Fellow of Lincoln College, Professor of Pathology
in the University of Oxford; and E. W. Aintey WALKER, D.M., Fellow
and Tutor of University College, Lecturer in Pathology in the University
of Oxford.
(Communicated by Prof. C. S. Sherrington, F.R.S. Received November 22, 1913,
—Read January 22, 1914.)
(From the Department of Pathology, University of Oxford.)
In the course of investigations on the production, distribution, and rate of
disappearance in the body of immune substances, we were occupied in 1908
and previous years with a series of experiments on agglutinins, and we
arrived at conclusions pointing to their close relationship to the blood and
blood-forming organs (1, 2). In association with these inquiries, one of us
(G. D.), together with W. Ray, published a communication on the relation-
ship between the blood volume and the distribution of agglutinins within
the circulation (3).
It was there shown that the concentration of this substance (agglutinin)
in the blood after inoculation into an animal was proportional to the body
surface of the animal concerned, and was thus approximately proportional to
the two-thirds power of the weight. Hence was deduced the conclusion that
the blood volume of the animals examined was proportional to their body
surface. :
The recognition of this relationship between surface and blood volume
made it clear that the assumptions hitherto made use of in attempting to
determine dosage for the administration of therapeutic substances such as
antitoxins and drugs required complete revision, at any rate, in so far as
the activity of these substances might be supposed to be dependent on their
concentration in the circulating blood. Subsequently the surface relation
(W*) was taken up by B. Moore (4) in an interesting communication dealing
with the dosage of drugs, and especially with the therapeutic action of
atoxyl and various other compounds of the heavy metals in the treatment of
trypanosomiasis. Moore came to the conclusion that, in regard to these
drugs, tolerance was limited by the surface area of the mucous membrane of
VOL. LXXXVII.—B. 2A
320 Prof. G. Dreyer and Dr. E. W. A. Walker.
the alimentary canal. This, in his opinion, offered a valid explanation of the
fact that it is often difficult or impossible to administer effective doses of
these drugs to large animals, since these animals do not tolerate the doses
which would be required to produce the same concentration in their blood
as 1s needed for successful therapeutic action in small animals.
The observations on agglutinins had already led us to the conclusion that
the concentration of inoculated drugs and other foreign substances dis-
tributed in the blood plasma would necessarily be proportional to the
surface of the animal, since it was shown that the blood volume was always
proportional to the body surface. And this fact would equally apply within
any given species to an internal surface such as that of the alimentary canal.
The blood volume formula had, however, immediately and seriously been
called in question (5, 6). Accordingly, it was necessary to turn aside from
the problems in hand until the criticisms offered had been carefully
examined, and the relationship of blood volume to the surface area had been
adequately confirmed. This, so far as we are able to judge, has now been
done (7).
Accordingly, we now proceed on the assumption that the blood volume of
warm-blooded animals is a function of their body surface, and is given by the
formula B = W”/k, where n is approximately 0°72, and i is a constant to
be ascertained for each particular species of animal.
Using this assumption, we find that the minimal lethal dose of a long
series of substances of widely different origin and composition can satis-
factorily be expressed as a function of the body surface. The series of
substances which have been found to follow this law includes not only
organic bodies both of animal and vegetable origin, but also a number of
inorganic compounds,
It follows that we are entirely in accord with the main conclusion reached
by Moore in his very suggestive discussion of the subject, namely, that
dosage must be proportional to body surface (in warm-blooded animals).
But, in view of the results which follow from the application of the blood
volume formula, we are unfortunately not in a position to agree with the
line of argument by which he deduces this conclusion from a consideration
of the area of the alimentary tract. Moreover, we are quite unable to admit
as satisfactory the explanation which he offers of the fact that drugs which
are successful in the treatment of various species of small animals are not
successful in the case of species of large size in any dose which can be used
with safety. For reasons which will be given below, the explanation of
these facts appears to us to be dependent on a specific tolerance or
intolerance, as the case may be, in different species of animal, and not upon
Muumal Lethal Dose of various Torre Substances. 321
the difference in their size or relative area of alimentary surface as suggested
by Moore.
In proceeding to discuss our own examination of the subject of the
present communication, we begin by endeavouring to show how the dose of
a given poison which kills animals of a particular species in a given time is
related to the weight of the individual.
For this purpose certain experiments with diphtheria toxin, the full
details of which will be found in a subsequent paper (“An Analysis of the
Problem of the Minimal Lethal Dose, ete.”), are made use of, and some of the
results obtained are given below.
In the case of diphtheria toxin, if the death time (34 days) which is
usually taken in the standardisation of this substance be made use of, it is
found that for guinea-pigs which die in about 80 hours the lethal dose
expressed as a percentage of the weight works out as follows :—
For animals of between—
200 and 250 grm. weight, about 6.0 cu. mm. of Toxin B per 100 erm.
310 ,, 370 5:2
»” »”
415 ,, 530 7 < 5:0 » » »
Again, taking a death time of some 40 hours, it is found that in the
lightest group of guinea-pigs the minimal lethal dose per 100 grm. of weight
is about 6°5 cu. mm. of the toxin, in the group of medium weights it is about
6:2 cu. mm., while in the heaviest group it is about 6 cu. mm.
Hence it follows that, for individuals of differing weights, the minimal
lethal dose cannot be rightly expressed as a percentage of the body weight.
This fact is, of course, well known to those familiar with the routine
estimation of toxicity.
In Tables I and II are given two groups of animals, the one group
consisting of light individuals and the other of heavy ones; where the
dosage expressed in per cent. of body weight was approximately the same.
The average weight of the animals in Table I is 234 grm., the average dose
per 100 grm. is 6:3 cu. mm., the dose estimated in relation to the surface area,
and calculated from the expression D = d/W°™, where dis the actual quantity
of the drug introduced, is 29:1 cu. mm., and the average time to death is
46 hours.
The average weight of the animals in Table ILis 425 grm., the average dose
per 100 grm. is again 6°3 cu. mm., but the dose calculated in relation to the
surface area has increased to 34:3cu. mm. and the average time to death is
seen to be reduced to 37 hours. It is, therefore, evident that when the
dose per 100 grm. of weight is made the same in light and heavy groups of
B22 Prof. G. Dreyer and Dr. E. W. A. Walker.
Table I.—Experiments with Diphtheria Toxin B in Guinea-pig (subcutaneous
fo)
injection).
Group of Light Animals.
] | |
: | Dose (D) in | | Dose in per
GOS Nal Weekes Actual dose | relation to sate. | cent. of weight
No. | of experi- of animal, 1 j Pace x 107 of hours to 4
ment Fegeony d,ine.c. | surface x UE | demah cu. mm.
: 5 1D) = GN, | ° per 100 grm.
|
Cee a ee ama
1 44 215 0 01400 2940 42 6°51
24) SHAS. Pe E280) 0°01375 | 2745 64 5°99
a | 42 255 O-01575 | 2920 40 | 6°19
A | 45 235 0:°01585 =| 3020 38 6°53
as Aa — :
Average......... 234 | 0-01471 2906 46 6°31
Table [1—Experiments with Diphtheria Toxin b in Guinea-pig (subeutaneous
injection).
Group of Heavy Animals.
| ; Dose (1D) in Dose in per
y o 2 _
N f Nc: ‘ A cien Actual dose relation to of ees hs cent. of weight,
Sle ee bl lees ed TN Gh, Tht 0. surface x 107 ee, cu. mm,
a ee ae aa D = dfwr?. | : per 100 grm.
1 28 425 0 ‘02780 3560 30 6°55
; 2 32 415 0 :02715 3525 49 6°54
3 31 435 0 02845 3580 30 6°54:
4 35 415 0 °02480 3220 44, 5°98
| 5 30 435 0:02590 | 3258 32 5°96
| a
Average......... 425 0:02682 | 3429 37 6°31
animals of the same species the lighter animals survive for a much longer
period than do the heavier. The explanation of this difference in death
time is to be sought in a comparison of the doses calculated in relation
to the surface. It is then seen that the dose thus calculated is much
smaller in the lighter animals than in the heavier group.
That this is a valid method of calculating dosage follows from the fact that,
under ordinary conditions, substances administered as drugs, to act after
absorption into the body, must become diluted in proportion to the volume
of the blood. They are carried to the tissues of the body through the
medium of the plasma in a relative concentration which is determined by
the volume of the circulating blood. But the volume of the blood is a
function of the body surface. Accordingly, it follows that the concentra-
Minimal Lethal Dose of various Toxic Substances. — 323
tion in the plasma of any substance administered to animals under like
conditions in doses proportional to their body weights will be much less
in the lighter animals than in the heavier individuals of the same species.
On the other hand, if the doses be administered in relation to the body
surface, their initial and their maximal concentration in the plasma will
be the same whatever be the weights of the individual animals concerned.
The results brought forward for diphtheria toxin do not constitute an
isolated instance in support of this view, that in any given species of
animal dosage must be used in relation to the volume of the blood. Very
numerous observations from the literature of toxicology which we have
collected and analysed confirm the accuracy of this method of measure-
ment. It appears to hold, so far as we have been able hitherto to ascertain,
for a large number of substances of very different constitution and of
diverse mode of action in warm-blooded animals. Wherever a sufficient
number of accurate data can be found the effect of dosage can be shown
to be related to blood volume and surface area in any given species.
Numerous results which have been thought to be inexplicable when the
dosage was expressed in per cent. of body weight, except on the ground
of special individual susceptibility or individual resistance, in reality give
precisely the results which would have been expected had the dosage been
expressed in terms of body surface.
In the case of arsenic (As203) in the rabbit the observations of
Morishima (8) afford an interesting illustration. The data and the calcula-
tions from these observations are given in Table III. Here it is seen that
the time of death shows no exact relation to the dose expressed in per
cent. of weight, but it follows quite closely the dose in relation to surface,
though animal 5 shows an irregularity im living longer than animal 4.
It will, however, be seen that the average dose per surface of animals 3
and 5, taken together, and their average time of death are identical with
Table I1].—Arsenic (As2Q3) in Rabbit, Morishima’s Experiments
(subcutaneous injection).
| | P | Dose (D) in | Dose in per
| No. | Te une | relation to SEER, of pie, te cent. of eights |
Wie se car ; orm. | in mgrm. cea mgrm.
| | 8 PD aiwer! per 100 grm.
| i H
| | 7 a Aine |
eal 1324 8°61 4,86 ee) 0°65
2 1103 7°72 4°95 eo 0°70
3 | 1495 10 ‘47 5 AQ 84, 0-70
4 1112 8 90 5 67 96 0°80 |
By | 1702 11 ‘90 5) “Gil 108 0°70
324 Prof. G. Dreyer and Dr. E. W. A. Walker.
the surface dose and time of death of animal 4, while the doses in per
cent. of weight differ by 14 per cent. Again, if we compare the three
animals which received equal doses, if the dose is expressed in per cent.
of weight (viz., 2, 3, 5), the lightest one survives while the two heavier
ones succumb. The explanation is at once evident on comparing the size
of the doses expressed in relation to surface.
In Table IV is given another series of observations by Morishima where
the injection was made intravenously. This method is, of course, likely to.
yield more precise results than subcutaneous inoculation, and it is seen that
the effects of the doses, when the latter are expressed in relation to body
surface, are remarkably regular and striking. On the other hand, when the
dose is given in percentage of body weight, as was done by Morishima himself,
the time to death varies very widely in animals which received equal dosage
with the drug on his method of calculation. Morishima’s results might be
taken to indicate great individual differences in susceptibility in different
individual animals. But such individual differences do not appear when the
dose is calculated in relation to blood volume and body surface.
Table I1V.—Arsenic (As203) in Rabbit, Morishima’s Experiments
(intravenous injection).
ae Dose (D) in | Dose in per
N W eee Actual dose | relation to surface, ies | cent. of roette |
o. | of animal E | : of hours to | |
5 , (d), in mgrm. | in mgrm. death mgrm. |
| ai | D=d/Wee, : per 100 grm. |
!
| el 1135 6°81 4°30 | 20 0 60
2 1190 7°73 4°71 432 0°65
3 970 6°79 4°81 | 48 0-70 |
4 1155 8 08 5 04 21 0-70 |
5 1952 13 66 5 82 8 0°70
Similar facts can readily be made out from various other experiments which
have been carried out with arsenical compounds by a number of observers.
In the case of another heavy metal, cobalt, the same results hold good
when the dosage is expressed in relation to surface instead of in the usual
manner as a percentage of the body weight. This fact has been determined
by an analysis of Meurice’s experiments (9) on pigeons injected into the
breast muscles with cobalt nitrate, Co(NOs3)2 This is of special interest in
view of the fact that it has already been shown in the experiments which we
carried out in association with H. K. Fry, referred to elsewhere (10), that
the blood volume of birds (like that of mammals) is proportional to their
surface area.
Minimal Lethal Dose of various Toric Substances. 325
Without further multiplying detailed instances it may be stated that we
have obtained the same results on calculation from a variety of published
observations on a number of different substances administered by various
methods in different animals. Among these substances are sulphate of methyl
brucium injected subcutaneously in- the rabbit, and codeine hydrochloride
administered by the stomach in the same animal (Crum Brown and Fraser
(11) ); sulphate of physostigma given subcutaneously in rabbits (Fraser (12) );
morphia and atropine sulphate administered subcutaneously in the rat
(Bashford (13) ); various snake venoms—krait, Enhydrina valakadien, Enhy-
dris curtus, cobra—inoculated in rats, rabbits, guinea-pigs, and cats by
different observers (Fraser and Elliott (14), Elhott, Siller, and Carmichael
(15), Madsen and Noeuchi (16), and others); adrenalin both natural and
synthetic in the mouse (Schultz (17) ); tetanus toxin injected subcutaneously
in the mouse (Knorr (18)); potassiwm chloride (KCl) introduced intra-
venously in the rabbit (Hald (19) ); and caffeine subcutaneously, intraperi-
toneally, intravenously, or by the mouth in dogs, cats, rabbits, and guinea-pigs
(Salant and Rieger (20) ).
In view of the conclusions to which the results obtained with all these
very diverse toxic agents lead, it seems clear that in animals of different
size in any given species the dose required to produce a given effect is related
to the surface and blood volume of the animal and not directly to the body
weight. That is to say, the smaller individuals require a relatively larger
dose than the heavier animals to produce the same effect.
While we are not prepared to maintain that this constitutes a universal
rule to which there are no exceptions, yet it follows from what has been
already stated that it possesses a very wide application, and we have not up
to the present met with any exception in the case of mammals and birds.
Accordingly we conclude that if it is desired to administer comparable
doses of drugs in warm-blooded animals of different size and weight in any
given species, they must be calculated in relation to the body surface.
It follows from this that if one administers any given toxic substance in
doses such as will kill each of the animals employed in about the same period
of time, one is now in a position to use animals of various size over a wide
range of weight within the same species instead of only animals of one
particular size in carrying out experimental work upon toxicity and lethal
dosage. One is no longer restricted to the use of carefully selected animals
of a given and standard weight, as has hitherto been the case, for example, in
all determinations of the strength of toxins as well as in the standardisation
of antitoxins. This result will naturally prove of value in facilitating
toxicological investigation in very many directions.
326 Prof. G. Dreyer and Dr. E. W. A. Walker.
In case of cold-blooded animals we are not at present able to put forward
any definite statement ; but the problems which they present are under inves-
tigation.
As regards the influence of sex in warm-blooded animals, we find an
indication in our figures that female animals require a somewhat smaller dose
to produce a given effect than male individuals of corresponding weight.
This agrees with what has frequently been pointed out as the result of
clinical experience. The observation seems to find its explanation in the fact
that the blood volume of female animals is slightly smaller(7) than that of
males. For both the initial and the maximal concentration im the plasma of
any. drug administered by a given route in a series of animals of different size
and weight will naturally be related to the volume of the plasma. Whatever
be the rate at which it is selected from the plasma by particular cells, and
whatever be the rate of its elimination from the body, the concentration in
the blood plasma of any given substance must at every stage be related to the
volume of that plasma in the individual animal concerned. Thus a given
dose of any substance administered Gn one and the same dilution) will reach
a higher effective concentration in those individuals whose blood volume is
less than in those in which it is greater.
The importance of this question of concentration may be illustrated by _
a reference to the experiments carried out by Hald (19) with potassium
chloride. These showed that in individuals of equal weight the effect of one
and the same dose of the active substance was greater, and manifested itself
more rapidly, the higher the concentration in which it was given.
In view of these considerations it becomes of interest to return to the
question of the failure encountered in the treatment of trypanosomiasis in
large animals. with drugs successfully employed in the smaller species.
If one compares the doses necessary to produce the same concentration of
a given drug in the plasma of man and the rat, it can readily be shown that
even if a man of 70 kerm. could be given the same dose per kilogramme as a
rat of 140 grm. weight—the figures selected by Moore in his discussion—
the concentration of the drug in the man’s blood plasma would only be about
75 per cent. of that obtained in the rat. Accordingly, the same therapeutic
effect could not be produced. Man, however, cannot tolerate anything ap-
proaching this degree of dosage, and hence the treatment which is curative
in rats becomes inapplicable in the human subject. But even these facts do
not, as it seems to us, afford the whole explanation of the difficulty in question.
For it appears that differences in tolerance and intolerance to particular sub-
stances in different species of animal are of a specific character and cannot be
explained merely by relative differences in blood volume and body surface.
id
Minimal Lethal Dose of various Toxie Substances. 327
In proof of this, attention must be drawn to the fact that it is not always
the larger species which are more susceptible than the smaller species to
dosage proportional to their relative body surface, or even to their relative
body weight. Sometimes the conditions are reversed. Thus, as is well
known, a horse infected with tetanus may be found in apparently excellent
condition and as yet exhibiting no symptoms of the disease at a period when
its blood already contains enough tetanus toxin to kill a guinea pig injected
with only a few cubic centimetres of the horse’s serum. Similarly in the
case of rats and guinea pigs, rodents of about the same size, the rat can resist
several hundred times the dose of diphtheria toxin which will be fatal to the
guinea pig within a few days.
In the case of substances other than bacterial toxins similar examples
showing a greater resistance in the larger species than in the smaller can
readily be found, as for instance in the experiments of Meurice, already
referred to (9), in Bock’s experiments (21) with cobalt compounds, in
Jodlbauer’s paper on Tetramethyl ammonium chloride (22), in Fraser and
Elliott’s experiments on Cobra venom and on Enhydrina venom (14), and
in many other pharmacological investigations. It follows that drug suscep-
tibility is by no means necessarily greater in the larger species than in the
smaller, but on the contrary it is frequently less. Accordingly, any general
explanation of drug action in different species of animals, which is based upon
the relative size of their surface, cannot be maintained. Only within one and
the same species of animal will the surface relation prove a reliable guide in
dosage. ,
In this connection it is of some interest to consider briefly formulee for
dosage in the human subject such as have been made use of or suggested by
various writers. For the sake of ease in calculation these have usually been
based on the age of the patient, and most of them appear to aim at giving
dosage in relation to the body weight.
But in the case of the formula of Thomas Young, 12815, we meet the earliest
example of dosage calculated so as to give younger individuals a relatively
greater dose per unit of body weight than is given to adults. Young wrote
that “for children under twelve years old, the doses of most medicines must
be diminished in the proportion of the age to the age increased by twelve :
for example at two years old 1/7 = 2/(2 + 12). At twenty-one the full dose
may be given. Y.” (23).
We owe the exact reference to the kindness of Dr. A. J. Jex Blake; but
how Young arrived at his formula, Age/(Age + 12), it has not been possible
to discover from his writings. However this may be, his formula actually
gives for all ages from about four or five to about 16 a dosage fairly
VOL, LXXXVII.—B. 2B
328 ‘Prof. G. Dreyer and Dr. E. W. A. Walker.
approximating to dosage by the surface area. But below the age of five years
the dosage by Young’s formula falls more and more rapidly below the dose
calculated in relation to body surface.
We append a table showing the doses which would be given’at different
ages from 1 year to 21 years in a system of dosage calculated in relation to
blood volume and body surface, taking the weights at the different ages as
given in Vierodt’s tables, 1893.
Table of Dosage,
| |
Age, | Average weight, Dose in relation to | Dose as a fraction
in years. | in grm. surface. | of dose for adult.
21 61,200 100-0 1
20 59,500 98 °8 |
19 | 57,600 95°7
18 | 53,900 92°5
17 49,700 86-2
16 | 45,400 81-4
15 41,200 75:1 $
14. 37,100 70-1
13 33,100 64°7
12 | 29,000 58°38
11 27,000 55-4 .
10 | 25,200 52°8 |
9 | 23,500 50°6 | 2
8 | 21,600 47-2
7 19,700 44.6
6 | 17,800 41‘1
5 15,900 38-1
4 | 14,000 34-3 a
3 12,500 Bil 7
2 | 11,000 29°2
1 | 9,000 25:1 a
0 3,100 11°8 | aw
In the abovejtable the dose in relation to the surface is given as calculated
fromthe body weight,and pointsareindicated where thecalculated dose approxi-
mates to a simple fraction of the adult dose. These work out extremely
conveniently for practical application, Thus at 15 years (approximately
three-quarters of the adult age of 21) the dose is #?; at 9-10 years
(nearly half the adult age) the dose is $; at 3-4 years it may be given as 4;
at 1 year of age it is };- below that age it sinks to as little as =4, of the adult
dose.
In conclusion we would draw attention to the fact that as long ago as
1818 Hufeland (24) gave the dose at fifteen years as ? and the dose at one
year as }, though heiplaced the half dose at six years of age instead of at
nine or ten as our table makes it. Thus it appears that both he and Young
already recognised the necessity of giving relatively larger doses than
Mimmal Lethal Dose of various Toxic Substances. 329
would be proportional to body weight in the younger and smaller individuals
of the species. On the other hand Dilling (25) in a recent communication
proposes a formula which approximates roughly to a dosage per kilogramme of
body weight. This is a system of dosage the fallacy of which was emphasised
by Moore. In view more especially of the considerations brought forward
above, we venture to suggest that it should now be entirely abandoned.
Conclusions.
1. In warm-blooded animals of the same species but of different weights
dosage must be calculated in relation to the body surface.
This result agrees with the conclusion already reached by Moore* though
on different grounds.
2. This statement is to be explained on the ground that the concentration
in the plasma of any given substance administered is dependent on the
volume of the circulating blood, which is itself proportional to the body
surface in any given species of animal.
3. It follows that in the accurate measurement and standardisation of
toxic substances and antitoxins it will now be possible to make use of animals
of different weights within a given species instead of using only animals of
an arbitrarily selected weight, as has hitherto been necessary.
4. Results in dosage calculated from one species of animal cannot
directly be apphed to another species merely by taking surface into
due consideration, since tolerance and intolerance are specific characters
which are shown to be in many cases independent of the size of the species
concerned.
_ 9. For the human subject dosage in relation to the surface works out very
simply as approximately :—
ENTAIL GR Soe Full dose AG. 34 YCa TS esa eeee eee + dose
” 15 99 tee eee eee 3 ” ” I PLUM ey ve 9 cl ele td 4 ee
PO NO ste: ene: BS a, In the early months ... +4 ,
REFERENCES.
1. Dreyer, Georges, and Walker, E. W. Ainley, “Observations on the Production of
Immune Substances,” ‘ Journ. Pathol. and Bacteriol.,’ 1909, vol. 14, p. 28.
2. Dreyer, Georges, and Walker, E. W. Ainley, “On the Difference in Content of
Agglutinins in Blood Serum and Plasma,” 7bzd., 1909, vol. 14, p. 39.
3. Dreyer, Georges, and Ray, W, “Observations on the Relationship between Blood
Volume and the Total Amount of Agglutinin recoverable from Actively and
Passively Immunised Animals,” zb7d., 1909, vol. 13, p. 344.
* Moore’s conclusions seem to rest in the main on a consideration of “ substances
which act by stimulation or inflammation of surfaces.”
330
4,
or
~I
9.
10.
18.
19.
20.
21.
22.
~ 23.
24,
25.
Minimal Lethal Dose of various Toxic Substances,
Moore, B., “‘ The Relationship of Dosage of a Drug to the Size of the Animal treated,
especially in regard to the Cause of the Failures to cure Trypanosomiasis and
other Protozoan Diseases in Man and large Animals,” ‘ Biochemical Journal,’ 1909,
vol. 4, p. 323.
Chisolm, R. A., “On the Size and Growth of the Blood in Tame Rats,” ‘Quart.
Journ. Exper. Physiol.,’ 1911, vol. 4, p. 207.
Boycott, A. E., ‘The Size and Growth of the Blood in Rabbits,” ‘ Journ. Path. and
Bacteriol., 1912, vol. 16, p. 485.
Dreyer, Georges, Ray, W., and Walker, E. W. Ainley, “On the Blood Volume of
Warm-blooded Animals, together with an Inquiry into the Value of some Results
obtained by the Carbon Monoxide Method in Health and Disease,” ‘ Shee
Archiv f. Physiol.,’ 1913, vol. 28, p. 299.
Morishima, K., “Giftigkeitsgrad, Absorptionsgeschwindigkeit und Immunisirungs-
vermoégen des Arseniks,” ‘ Archives Internat. de Pharmacodynamie et de Thérapie,’
1900, vol. 7, p. 65.
Meurice, J., ‘Intoxication et Désintoxication de différents Nitriles par /Hypo-
sulfite de Soude et les Sels Métalliques,” ‘Archives Internat. de Pharmacodynamie
et de Thérapie,’ 1900, vol. 7, p. 11.
Dreyer, Georges, and Walker, E. W. Ainley, “The Effect of Altitude on Blood
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et de Thérapie,” 1901, vol. 9, p. 451.
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331
Experiments on the Restoration of Paralysed Muscles by Means of
Nerve Anastomosis.* Part Il.—Anastomosis of the Nerves
supplying Limb Muscles.
By Rosert Kennepy, M.A., D.Sc., M.D., St. Mungo Professor of Surgery in
the University of Glasgow.
(Communicated by Prof. J. G. McKendrick, F.R.S. Received November 25, 1913,—
Read January 22, 1914.)
(Abstract. )
The part of the research into the anastomosis of nerves dealt with in this
paper has reference to the restoration of function of a group of muscles in the
limb. Following the early experiment of Flourens, several workers (Rawa,
Stefani, Howell and Huber, Cunningham, and the author) published investi-
gations on the effects of nerve crossing, or the division of two nerves in the
limb and cross suture of the ends.
These investigations, while showing that restoration of function can take
place through the composite nerve, and that even in the cerebral cortex the
areas associated with flexion and extension become interchanged, left it
doubtful whether in the event of one nerve being eliminated, the muscles
supplied by it could be innervated by a neighbouring motor nerve, which at
the same time could continue also to supply the muscles proper to it,
performing thus a double function.
The subject was investigated from this point of view by Kilvington, who
published a series of experiments in which the external popliteal nerve was
cut and the peripheral segment anastomosed to the internal popliteal, and
vue versd. He reports recovery of function after this procedure.
Doubt, however, still remains as to the possibility of an extensor and
flexor group of muscles recovering the capacity of performing co-ordinated
movements under such conditions, as in the case of the hind limb of the dog
even when the sciatic nerve cut high in the thigh has not united, the animal
is able to use the leg in walking, the chief defect being that it walks on the
dorsum of the paw. Also, in these circumstances, the foot is sometimes,
possibly by accident, placed plantar surface down. Therefore the reported
recoveries after such experiments leave doubt whether the recovery is real or
apparent. In addition, it is impossible to investigate the changes in the
* The expense of this research has been defrayed by a Government Grant from the
Royal Society.
VOL. LXXXVII.—B. 2 ¢C
332 Prof. R. Kennedy. Restoration of Paralysed
cortical representation in the case of the hind limb, as in the dog this is
represented by a single centre.
The author has performed five experiments in dogs on the right fore-limb,
which has the advantage, in the first place, that section of the nerves above
the elbow paralyses the limb in such a way that it is impossible for the
animal to use it for walking until recovery of co-ordinated movements
occurs. ‘This is then a very severe test. In the second place, the fore-limb
of the dog is represented by two separate and distinct centres, one for flexion
and one for extension, which are only exceptionally defective.
The experiments are of two kinds, but are the same in so far that in all
the musculo-cutaneous, median, ulnar, and musculo-spiral nerves were each
divided above the elbow, and the limb thus completely paralysed below the
elbow. Then the four distal segments were united to the proximal end of
the musculo-spiral nerve, and the three remaining proximal ends left
ununited ; or, on the other hand, the four distal segments were united to the
proximal ends of the musculo-cutaneous, median, and ulnar, and the one
remaining proximal end (musculo-spiral) left ununited. The limb was fixed
in plaster of Paris as long as necessary.
The following results were obtained :—
A. Where the central supply was that proper to the flexor muscles
(musculo-cutaneous, median, and ulnar), the first sign of recovery of the
muscles was shown at 96 and 93 days respectively after the operation, and
a satisfactory recovery, enabling the dog to run about, and in one case to be
taken for exercise into the streets, was reached at 126 and 123 days respec-
tively. In the first case no interruption of the excellent recovery occurred
as long as the animal was allowed to live, namely, 225 days, but in the latter
case there was some trouble by the development of a slight flexor contracture
which hindered the recovery up till the animal was killed at 187 days.
B. Where the central supply was that proper to the extensor muscles
(musculo-spiral), the first sign of recovery was at 81 and 59 days respectively
in the two which showed recovery. In the first case a pressure sore inter-
fered with further progress, but in the second case at 79 days the animal ran
about normally, and. was able to be taken out for exercise in the streets.
without any fear of attracting notice. It may be noted that although in this.
form of experiment the muscles of the limb were supplied by the lesser
number of nerve fibres, namely, those contained in the musculo-spiral, the
recovery commenced earlier than when the three nerves normally supplying
flexors formed the sole central supply, although in that case: the greater
number of nerve fibres were available for the supply. This is explained as
due probably to the earlier recovery of the extensors when the musculo-spiral
Muscles by Means of Nerve Anastomosis. 333
was the common source of supply, thus enabling the leg to be earlier held
extended, and therefore useful for walking.
Each experiment was investigated before the animal was killed, as
follows :—
A. Exanvination of the Nerves.—In every case the union of the nerves had
taken place as intended, ic. no reunion of any nerve intended to be
eliminated had occurred. Also it was found that when the musculo-spiral
trunk was supplying both flexors and extensors, it conveyed the fibres for
the flexors and those for the extensors along different sides of the nerve,
where they could be separately stimulated.
B. Examination of the Brain—In every case in which this examination
was able to be made, namely, in one of the first type of experiment and
in two of the second, the centre at which stimulation normally produces
contraction in the muscles of the eliminated nerve supply was inexcitable,
and in the other centre (either the normal flexion or the normal extension
centre according to the type of the experiment) both flexion and extension
were evoked, and at no point in the centre could separation of these move-
ments be obtained.
The operation which Nicoladoni introduced for cases in which infantile
paralysis has destroyed the function of a group of muscles presents the
same problem as do cases of nerve anastomosis. This operation consisted
of substituting for the lost muscles a portion of a neighbouring muscle so as
to regain some of the lost function, and if the lost function or a part of it can
be thus regained, it can only be by the nerve supply of the muscle from which
the substitute is taken altering its function so as to cause the movement
proper to the paralysed muscle instead of that normally belonging to it.
A case in which the author performed this operation was carefully examined
in order to exclude sources of fallacy. The function of the extensors in the
leg was lost and a talipes equinus by contraction of the gastrocnemius was
present. This was in a girl aged 7, and had lasted for six years. The extensor
muscles gave no reactions to electrical tests. At the operation the gastroc-
nemius was lengthened to overcome the talipes, and one-third was taken from
the outer part of that muscle and attached in front to the tendons of the
paralysed muscles. Sixty-nine days after the operation the patient had
power to extend the foot voluntarily, some eversion being produced at the
same time owing to the line of action of the new muscle. The new muscle
also stood out as a tense band while the voluntary movement was being
performed. The two separate movements were also able to be evoked by
galvanic or faradic stimulation over the two separate parts of the gastroc-
nemius. After the recovery of voluntary extension a further examination
2c 2
334 Restoration of Paralysed Muscles by Nerve Anastomosis.
of the extensor muscles was made, and this showed that the movement of
extension was not made by the extensor muscles, as they were not able to
be stimulated. A platinum electrode was also inserted into the extensor
muscles through the skin, but no contractions could be produced in them
either by the galvanic or by the faradic current.
The following are the conclusions from the research :—
(1) In the limb of the dog when the nerve supply of one group of muscles
is eliminated, the nerve supply of its antagonistic group may be used to
supply both groups, and under these conditions co-ordinated movements may
be restored.
(2) When two antagonistic groups of muscles in the limb of the dog have
their nerve supplies cut and both groups then made to derive their supply
from that of the one group, the group whose nerve supply is utilised probably
will be the first to recover.
(3) Recovery of function of antagonistic muscles is slower to occur when
one nerve supply is eliminated than in the case of nerve-crossing experi-
ments where no nerve is eliminated, but where the supply of the two
groups is crossed: and this delay is caused by reduction in the former case
of the total volume of the nerves supplying the limb, and possibly by greater
difficulties of adaptation in the brain to the new conditions.
(4) Where in the dog one nerve has been made to supply not only its
own but also the antagonist of its own muscle, the nerve fibres passing to
the two muscles in the nerve trunk proximal to the junction may be so
completely separated that it may be possible to stimulate each group without
affecting the other, producing thus at will contraction either of the one or
of the other muscle, both being now supplied by a single central trunk.
(5) When two groups of antagonistic muscles in the limb of the dog are
represented by separate cortical areas, and when the nerve supply of one of
the groups is eliminated, both groups being caused to be innervated by the
remaining nerve supply, the cortical area corresponding to the eliminated
nerve supply becomes inexcitable, while the other cortical area on stimulation
causes contraction in both groups of muscles.
(6) Where one group of muscles is paralysed, and a portion of an
antagonist muscle is detached from its insertion and attached to the tendons
of the paralysed group, this substitute for the paralysed group may enable
the function of that group to be performed to a certain extent, and the
function recovered by means of this procedure is probably controlled by
the same adaptation in the central nervous system as occurs in the case of
nerve anastomosis.
(7) The adaptation in the central nervous system which allows restoration
Variations in the Sex Ratio of Mus rattus. 335
of function to take place after nerve anastomosis is not due to a simple
re-education process, as there is no evidence of this during recovery, but is
probably due to an alteration in the centres under the influence of altered
afferent impulses from the muscles, the brain thus having the capacity
quickly to adapt itself to such alteration.
Variations in the Sea Ratio of Mus rattus Associated with an
Unusual Mortality of Adult Females.
By F. Norman Wuire, M.D. (Lond.), Capt. I.MLS.
(Communicated by Prof. C. J. Martin, F.R.S. Received November 28, 1913,—
Read January 22, 1914.)
At the commencement of June, 1911, whilst engaged on plague epidemio-
logical observations in the United Provinces my attention was drawn to the
fact that nearly all the young Jus rattus that were being trapped and
examined by our staff in Lucknow were females. It was this strange
phenomenon, the parallel of which I had never encountered during a five
years’ experience of piague research in India, that prompted the inquiry,
the results of which are briefly set forth in this paper.
A few words explanatory of the methods employed in the daily routine
examination of rats will show the nature of the material at my disposal. The
prime object in trapping and examining large numbers of rats was, of course,
to ascertain how far facts thus collected would assist us in solving the plague
epidemiological problems with which we were faced. The species, sex, and
weight in grammes of each rat caught were noted; the address of the house in
which the rat was trapped and the number and species of fleas found on it
were recorded. The sex of each rat was noted after dissection of the animal,
and if it were female a further note was made as to the existence of
pregnancy. If pregnant the number of foetuses was likewise written down.
Finally, any pathological or other condition calling for comment was fully
described.
All this information, which was in the first place recorded on cards,
one card for each rat, was at the end of the day’s work entered in a
register. Weighing the rat was done in a specially constructed spring
balance, by means of which the weight in grammes to the nearest multiple
of 10 could be readily and accurately ascertained. I wish to emphasise
the fact that the sex of the animal was noted only after dissection, so that
336 Dr. F. N. White. Variations in Sex Ratio of Mus rattus
mistakes under this head were very unlikely to occur. A young female is
not always easily differentiated from a young male by external inspection only.
Under ordinary circumstances Jf, rattus would appear to be slightly
polygamous. Some observers have stated that it is very markedly so, and
explain the fact that about equal numbers of males and females are usually
caught by the alleged shyness of the adult female. This point I have
carefully gone into and believe the allegation has no foundation in fact.
As will be shown later, about equal numbers of males and females appear
to be born under normal conditions. When adult age is reached there is
either a somewhat enhanced mortality amongst males as compared with
females or else males are more wary and so less readily caught. It should
be noted also that females are as readily caught at the height of the breeding
season as at other times.
In the presence of severe plague a condition of marked polygamy is some-
times met with. I believe this to be chiefly due to the fact that plague is a
more fatal disease to male than to female rats.* When polygamy is marked
rats are scarce or difficult to trap; on the other hand, when the rat population
is very large the numbers of the two sexes trapped appear to be more nearly
equal. In support of these statements Tables II-V have been produced.
(1) Ballia district: Here plague is always present and rats, probably in
consequence, are very difficult to catch. Out of 4525 MZ. rattus caught
during 10 months 2550 were females, a proportion of 77 males for every
100 females. If the rats be separated into two groups, young and adult,
considering half the rats of 90 grm. and all those of lesser weight as young
and the remainder as adults, the degree of polygamy prevailing amongst the
rats of Ballia is seen to be even more marked than the above figures indicate.
Of the 1875 young rats 943 were male and 932 female, whereas of the
2650 adult rats 1618 were female and only 1032 male. In other words,
there were only 63 adult males for every 100 adult females (see Table V).
The rats were obtained from places scattered all over the district.
(2) Coimbatore Town: Has suffered from repeated but not very severe
epidemics of plague. J/. ratiws appears to be scarce; the catches were very
small. Here of 3889 M. rattws 2072 were females, 88 females for every
100 males (see Table IV). Here again the excess of females over males
affects adults only.
(3) Cawnpore: Here rats were extraordinarily numerous. When our
observations started a mild epidemic of plague was drawing to a close. No
acute rat plague was, however, met with.. During the previous few years
* See 3rd Plague Report, 1907, ‘Journ. Hygiene,’ vol. 7, p. 750, and 7th Plague Report,
1912, ‘Journ. Hygiene,’ vol. 12, p. 265, Table VII.
Associated with an Unusual Mortality of Adult Females. 337
the city had almost completely escaped plague, though in the more remote
past its epidemics had been extremely severe. Our very high catches
indicated a very large rat population and a complete recovery from the
onslaughts of plague. In Cawnpore of 51,181 J. rattws examined in one
year 25,838 were females, 7.¢., 98 males for every 100 females (see Table II).
(4) Banda Town has never suffered from epidemic plague. J. ratius was
present in very large numbers. In Banda of 10,127 rats caught during
11 months 5174 were females, 7.c., 96 males for every 100 females (see
Table ITT).
In Lucknow, in which city the very abnormal conditions, fully described
below, were present, of 34,908 I. rattus caught during the course of one
year 18,396 were females, 7.c.,89 males for every 100 females (see Table [).
Disturbance in the Sex Ratio of M. rattus in Lucknow and its Readjustment.
A reference to Table I will indicate the nature of the phenomenon in
Lucknow to which reference has been made. The table sets forth the
weight frequency distribution of male and female J. rattus respectively
for each of the 12 months from February, 1911, to January, 1912. There
appears to have been some influence at work destroying adult females and
sparing the males. This “influence” began its manifestations in March
and produced its maximum effect in May and June.
As if to compensate for the apparently wholesale destruction of adult
females, females only appear to have been born. These two processes, the
destruction of females and the suppression of male births, proceeded
pari passu. In June not a single male rat below the weight of 80 grm.
was trapped, whereas 610 females of less weight than 80 grm. were caught.
As the numbers of adults of the two sexes began to approximate more closely
the one to the other, young male rats were again trapped in increasing
numbers. In November, December, and January, the sex ratio approximated
to that normally pertaining.
Such, in brief, are the facts ; satisfactory explanations of the phenomenon
are difficult to come by. It may possibly be advanced that in the above brief
recapitulation of observed facts I have assumed more than the facts warrant.
The objection that is most likely to be raised is to the assumption that failure
to catch adult females signifies destruction of females. The shyness of the
female might account for the phenomenon. This point has been referred to, and
it was partly to meet this objection that I studied similar facts concerning
95,629 rats caught in various places. A reference to the tables of Cawnpore,
Banda, Coimbatore, and Ballia rats will show that in no place other than
Lucknow was such a circumstance observed. Female rats are not shyer or
338 Dr. F.N. White. Variations in Sex Ratio of Mus rattus
more difficult to catch than males ; on the contrary, it is possible that males
are slightly more wary than females. As stated above the two sexes are
normally produced in equal numbers, though adult females are usually in
slight excess of adult males.
It may also be objected that the parallel assumption, that failure to catch
young males signifies that no males are born, is not warranted by facts.
This is admitted, but the only other explanation that I can offer is that
the males were destroyed by their parents soon after birth (at a lesser
weight than 10 grm., when it becomes just possible to trap them with
the traps we employed, z.¢., at about a week old). That the parents should
have destroyed only the male offspring is, to me, less easy of credence than
that only females were produced.
It is a matter of regret that my observations did not succeed in throwing
any light on the causes of the female mortality. Plague was certainly present
until April, 1911, amongst the Lucknow rats, but it was not severe or wide-
spread, and, as has been pointed out, plague is more fatal to the male than to
the female. Whatever the cause was it was a widespread one in the city.
The rats, caught from all parts of Lucknow, represented as fair a sample as
could be obtained.
Further speculation on this interesting topic would not prove fruitful. The
rapid readjustment of the sex ratio after so grave disturbance is, to my mind,
the fact of chief interest. Im May and June when hardly any females were
produced there must have been an extreme degree of polyandry.
From a study of weight frequency curves of pregnant females, I have
concluded that for practical purposes 90 grm. represents a fair dividing line
between young and adult rats of Lucknow (Table VI). Half the rats of the
weight of 90 grm. can be considered young and half adult. Employing this
approximation, there appears to be an interesting correlation between the
excess of adult males over adult females and the excess of young females over
young males for the same month.*
In spite of the absence of any explanation of the facts the phenomenon
described appears to me to be of sufficient interest to warrant its publication.
* Mr. Major Greenwood, Jr., Statistician to the Lister Institute, has kindly supplied
me with the approximate correlations between the sex ratios of mature and immature
rats for the same month and also for certain sequences. The groupings appear in
Table VII and the coefficients in Table VIII. It will be seen that the negative
correlation for data derived from one month’s records is slightly larger than when the
records of successive months are combined. A possible explanation is that overlapping
of different affected colonies produces an apparent synchronism of cause and effect, but
the figures as they stand do not warrant any inference. The enormous excess of young
females in May and June is a statistically inexplicable fact.
339
Associated with an Unusual Mortality of Adult Females.
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340 Dr. F. N. White.
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341
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Associated with an Unusual Mortal
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342 Dr. F. N. White.
Table [V.—Coimbatore.
Variations in Sex Ratio of Mus rattus
{
January to March. April to June. July to September.
Weight in grms.
g 9 3 9 sua | 9 |
11-20 29 27 33 44 30 35
21-380 49 47 78 86 119 114
-40 62 43 50 51 72 74
-50 45 | 45 66 | 49 87 64
-60 32 33 48 48 76 78
—70 15 31 | 30 | 34 72 71
-80 18 30 29 49 52 66
-90 14 30 33 | 60 58 72
-100 1 11 36 51 63 46 94,
-110 13 23 21 57 33 80
-120 14 32 31 59 52 83
—130 18 16 21 | 29 40 56
—140 | 9 itil 23 | 15 37 38
-150 | Ce pal 7 16 14 20 27
-160 6 | 2 18 11 28 9
-170 6 2 15 7 27 10
-180 6 (0) 9 (0) 15 5
-—190 1 1 4 | 1 9 a
-200 1 1 | 1 3 1
-210 oft 2
—220 1 | 0
—230
—240 | |
-—250 |
|
362 | 416 577 678 878 978
Totals ve
W 778 1255 1856
Table V.—Ballia.—Consecutive rats caught between January 23 and
October 6, 1911.
Weight in grms.
11-20
21-30
-40
-50
-60
-70
—80
3 ?
102 82
182 171
147 139
127 135
98 116
127 109
110 113
100 134
127 155
89 | 168
128 222
102 248
103 224
| 74 182
M. rattus.
] 7 }
| Weight in grmms. é 2
160 74 142
170 67 89
180 49 51
190 51 34
200 58 23
| 210 34 9
| 220 21 1
230 4 2
| 240 1 0
250 0) 0)
260 0 0
270 0) (0)
280 (0) 1
i
Associated with an Unusual Mortality of Adult Females. 343
Table VI.—Lucknow.
M. rattus.
‘ . Number Number of Number of 3
Weight in grms. pregnant. ee. Per cent. ary Average.
51-60 6 W087 sie Ont | 31 orl
61-70 29 1026 2°58 135 4°6
-80 70 995 7 371 5°3
—90 120 917 13 614 5-1
-100 151 971 155 772 byail
-110 200 968 20 °6 | 1135 5°6
-120 303 1208 25 1670 5°5
—130 429 1648 26 2444 5°7
—140 423 1373 30°8 2509 5:9
-150 359 10384 84°7 2154 6
—160 292 778 87°5 1795 _ wal
-170 305 678 44, °8 2000 6°5
-180 1384 279 48 856 6°4
-190 101 187 54 657 6°5
—200 40 80 50 272 6°8
-210 21 39 53 °8 118 56
above 210 32 48 66 °6 217 6°8
Table VII.
| | ; | {
Weight in Weight in
a, 3 fe) Total. rs 8 | f°) Total. |
April. May-—April
90 or less 106 561 667 90 or less Chy q)) Byle/ 662
Beyond 90 943 681 1624 Beyond 90 943 681 1624
1049 1242 2291 1028 1252 2286
May. May—March.
90 or less 85 577 662 90 or less 85 577 662
Beyond 90 1180 599 1779 Beyond 90 984 948 19382
| 1265 1176 2441 | 1069 1525 2594
March. April—March.
90 or less 307 532 839 90 or less 106 561 | 667
Beyond 90 | 984 948 1932 Beyond 90 984 948 1932
| 1291 1480 2771 1090 1509 2599
344 Messrs. Hill, McQueen, and Flack. Conduction of the
Table VIII.
Source of Mature | Source of Immature | Coefficient of
Rats (over 90 grm.). | Rats (90 grms. or less). Correlation.*
March data March data —0°23+0:°04
April ,, April ,, —0°70+0 ‘02
May * May ,, —0°81+0°'01
April _,, May ~,, —0°76+0 :02
March ” May ”» —0°'69+0 "02
March ,, PAT YI, — 0°62+40 ‘02
* The coefficients were determined by means of Pearson’s approximate method (‘ Phil. Trans.,”
A, vol. 195, p. 16, equation lvii) and the probable error assumed to be 3[0 67449(1 —7”)//7].
The Conduction of the Pulse Wave and the Measurement of
Arterial Pressure.
By Leonarp Hitt, F.R.S., James McQUEEN, and Martin FLack.*
(Received December 20, 1913,—Read February 5, 1914.)
(From the Physiological Laboratory, London Hospital Medical College, London Hospital
Research Fund ; and the Pathological Laboratory, Aberdeen University.)
It is now well established that in cases of aortic regurgitation placed
in the horizontal position the arterial pressure is considerably higher
(50-80 mm. Hg) in the leg than in the arm.t Such pressures are taken by
the sphygmomanometer, using the armlet method, the armlet being placed
respectively round the calf and the upper arm, the disappearance and
reappearance of the pulse wave being noted in the dorsalis pedis or posterior
tibial artery, and in the radial.
In seeking for an explanation of this phenomenon it has already been
suggested by ust that the “lability ” of the arterial wall plays a considerable
part, the term “lability ” beimg used to designate the ease with which an
artery distends with a rise and recoils with a fall of arterial pressure. The
effect of increased and of diminished lability of the vessel wall upon the
conduction of the pulse wave has been demonstrated schematically by
* During tenure of Eliza Ann Alston Research Scholarship.
+ Hill, Flack, and Holtzmann, ‘ Heart,’ vol. 1, p. 73 (1909) ; also Hill and Rowlands,
‘Heart,’ vol. 3, p. 222 (1912).
{ Hill and Flack, ‘ Roy. Soc. Proc.,’ B, vol. 86, p. 365.
Pulse Wave and the Measurement of Arterial Pressure. 345
‘Russell Wells* on rubber tubing made with a thickness of wall corre-
sponding to an artery. While the lability effect has been shown by us in
exposed arteries, in the body the main arteries are surrounded with tissues
permeated with small arteries into which the blood pulsates. As the
arterial wall is supported by the pulsing tissues the lability effects obtained
on the exposed arteries cannot be directly ascribed to the same arteries
in situ. Further investigation must be made on these.
Now J. McQueen, Ingram, and Leonard Hillt have shown that there is
an extraordinary difference in the pressure required to damp down the
pulse wave in arteries such as the aberrant radial and the dorsalis pedis,
where these run superficially and lie upon bone, as compared with the same
or other arteries lying in the midst of pulsating “resonating” tissues.
They suggest that the pulse wave is supported on its way to the periphery
by the pulsing tissues, and that the higher leg readings obtained in cases of
aortic regurgitation may be due in part to the better conduction of waves
which have a high crest through the pulsating mass of the abdomen and
thigh. The reading of pressure in the case of the aberrant radial or dorsalis
is taken with the Leonard Hill pocket sphygmometer. The small bag of
this instrument when pressed on the radial artery (embedded in the tissues
of the forearm) gives the same readings as the armlet method. When
pressed on the aberrant radial or on the dorsalis pedis a far lower reading is
obtained, e.g.,1n a youth 35 mm. Hg against 110 mm. Hg.
We have constructed a wooden C-shaped box in which the arm can be
suspended freely by a sling. If the armlet be placed round this box so that
it presses on the front of the forearm, the obliteration of the pulse in the
radial is obtained by the same pressure as is required if the armlet be used
in the ordinary way. If the forearm be put in the box with the radial
border uppermost, and the aberrant radial be pressed upon by the armlet,
then the pulse is obliterated by a pressure of 35 mm. Hg. Using the armlet
in the usual way the pulse is obliterated in this artery by 110 mm. Hg.
In the one case the artery lying on bone is deformed by the armlet just as
it is deformed by the bag. In the other case the pulse in the aberrant radial
is not obliterated until the systolic pressure in the tissues of the forearm is
overcome.
We have recently investigated several cases of “ high blood-pressure ” and
find the following divergence between the readings of the leg and arm
arteries, using the armlet and the dorsalis pedis, the patients being in the
horizontal posture :—
* Russell Wells and Leonard Hill, ‘ Roy. Soc. Proc.,’ B, vol. 86, p. 180.
+ J. McQueen, Ingram, and Leonard. Hill, ‘ Roy. Soe. Proc.,’ B, vol. 87, p. 255 (1913).
346 Messrs. Hill, McQueen, and Flack. Conduction of the
Arm, | Leg, Dorsalis, L.H. | R is
armlet. armlet. small instrument. | ces
mm. Hg.! mm. Hg. | mm. Hg. |
G 225 | 275 139 Myocardial failure.
G’ 250 295 130 3 » (aortic regurgitation
| (Temporal 135) found post mortem).
| & 215 260 | 130 Myocardial failure.
(ee) 185 255 | 100 a i
|. | dat 130 215 45 Aortic regurgitation.
M1 180 175 65 Chronic nephritis.
M2 170 175 55 : »
Cl 105 175 80 Mesaortitis. |
In a normal individual we have found also that the variation in pressure
found on changing from the horizontal to the vertical position fully accords
with the effect of gravity, and that this is so when the readings are taken
from the dorsalis pedis or with the armlet round the leg. The dorsalis
reading in the vertical posture is increased by the gravity pressure just
as much as is the leg reading.
The divergence in readings between the artery lying in tissues or
exposed lying on bone has been fully substantiated by us in animals. If
we place round the neck of a dog an armlet connected with a recording
manometer and at the same time record the blood-pressure in the carotid
artery by a v. Basch C-spring manometer, we find that the pressure required
to obliterate the pulse wave on the tracing of the C-spring is just about the
same as the actual systolic blood-pressure. To graduate the C-spring we
~< «x
C SPTING
L7€CSSUTE
or armel
S$ pt tt yt tt ht
Fic. 1.
Pulse Wave and the Measurement of Arterral Pressure. 347
connect it with the Hg manometer and force up the pressure till the writer
of the C-spring reaches the crest of the pulse curve (fig. 1).
We obtain similar results on applying to the carotid artery (i sitw)
of the cat the bag of the small Leonard Hill instrument instead of the
armlet.
On the other hand, if we place a long length of the exposed carotid artery
on the convex surface of a watch-glass, we find that the pulse wave is
obliterated by a pressure in the bag much less than before. For example, in
a dog with an arterial pressure of 190 mm. Hg. it was found that with the
armlet 190 mm. pressure was required ; in the same animal with the artery
exposed but 60 mm. Hg. was required. In acat we found—
The systolic pressure was.............+ 65 mm. Hg.
And with the artery unexposed ...... 64 » Was necessary to damp
down pulse.
Artery lying exposed on muscles .... 26 is 55 S
Artery lying on scalpel handle (cf 12 3 8 f
fig. 2B).
To elucidate the cause of this marked difference in readings we devised
the following experiment :—A long length of cat’s carotid was exposed, the
uppermost part ligated and divided. ‘This end was first passed through a
T-piece and then an arterial cannula inserted into it, which in its turn was
connected with the C-spring manometer. As the artery passed in and out
of the T-tube through a piece of rubber tubing, the latter could be constricted
<_<
=190mm.Hg
tn T tube
Artery exposed on cover ylass
ey ama Lg.
Fia. 2A.
VOL, LXXXVII.—B,
348 Messrs. Hill, McQueen, and Flack. Conduction of the
so as to prevent any considerable leak of fluid from the T-tube. The artery
in the T-tube was then surrounded by Ringer’s solution and the pressure of
this raised. Obliteration of the pulse wave occurred when the pressure in
the T-tube reached that in the artery—190 mm. Hg. (fig. 24). The same
exposed artery removed from the T-tube and placed across the dome of a
watch-glass required but 27 mm. He (fig 28).
This experiment demonstrates the fact that the deformation of the arterial
lumen is the prime cause of the obliteration of the pulse wave. When the
artery is circularly and equally compressed by surrounding fluid, the pulse
continues to come through until the full systolic pressure is overcome. In
the case of the artery exposed and lying on a rigid surface, when the bag is
pressed on it the arterial wall is pushed in above and bulged out at the sides.
The lumen is thus converted from a circle into an ellipse, and resistance is
offered to the pulse by the changed shape. The force of the pulse is spent
on the labile wall of the artery in front of this resistance.
In corroboration of this experiment we have also found that if the finger
be gently laid along the course of the radial artery and the bag of the
sphygmomanometer pressed upon the finger until the pulse ceases to come
‘through under it, a less pressure is required than without such interposition
of the finger. This.is because the finger brings about the deformation of the
artery more easily than the bag. If the armlet be used and the finger be
inserted under the armlet to palpate the artery, one finds that the pulse
does not cease to come through under the finger until the full systolic
pressure is reached. Thus the readings were 35 and 97 mm. Hg respectively
in the case of a youth.
In the aberrant radial artery the pulse was obliterated by 55-60 using the
bag, by 35 using finger and bag. Using armlet and finger the radial was
quiterted by 135 and using armlet alone by 139.
A thin, distensible rubber bag inflated with a pressure of air can gage be
deformed from the spherical to elliptical or other shape without altering
the internal pressure. The bag may thus be made to take a shape which
would give great resistance to the passage of a pulse wave or flow of fluid,
although the total volume and pressure of the air in the bag remains
unaltered.
In the experiments on animals (goat, dog, cat) it was found that the
pressure required to obliterate the pulse wave in the exposed artery varied
from 25 to 60 mm. Hg. To elucidate the cause of the higher and lower
readings we measured the pressure necessary for the obliteration of the
pulse in the same exposed artery with the animal (cat) in the head-down,
horizontal, and feet-down position. We found that the pressure necessary
Pulse Wave and the Measurement of Arterial Pressure. 349
to obliterate the pulse varied markedly with the diastolic pressure within
the artery :-—
|
Diastolic pressure. | Obliteration pressure.
|
mm. Hg. mm. Hg.
Head-down......... 160 48
Horizontal ......... 134 28
Feet-down ......... 125 20
. |
The lability of the wall also plays a part, since with the same diastolic
pressure a higher pressure is required to obliterate the pulse in the carotid
artery of the dog or goat than in that of the cat.
McQueen, Ingram, and Leonard Hill found that when the pulse in the
aberrant radial artery was obliterated by a pressure in the bag of, say,
45 mm. Hg, the blood still trickled slowly into the artery. We have made
a cut into the exposed carotid of a cat and found that a pressure of
26 mm. Hg stopped the visible flow; a pressure of 20 mm. Hg allowed slow
oozing from a very elliptical lumen ; while a pressure of 10 mm. Hg allowed
the blood to spout freely through the incision.
In the above investigations the remarkable fact comes out that the pressure
required to deform the artery and to obliterate the pulse wave is considerably
below even the internal diastolic pressure of the vessel. To investigate this
phenomenon further, we compared the effect of perfusing, with the same
pulsating head of pressure, thin rubber tubing (about 0°7 mm. thick) and a
length of human artery of approximately the same calibre and thickness of
wall, and noting the external pressure required to obliterate the pulse wave
The results are as follows :—
4 Obliteration of pulse,
Systolic pressure. pressure in bag. |
|
mm. Hg. | mm. Hg.
Rubbers 225.-420-2- 140 195 (fig. 3)
Artery ...ec cece 130 46 (fig. 4)
The rubber tube resisted
deformation easily.
deformation, the labile arterial wall suffered
iN)
350 Messrs. Hill, McQueen, and Flack. Conduction of the
HH
WL
Meh Lh,
\
Rubber
| Pressure in
\ small bag
Geese
Oe Gobis
Artery
Pressure in
small bag
Fia, 4.
We then immersed a piece of the same rubber tubing in xylol. The tubing
quickly imbibed xylol and became swollen, less elastic, and more easily
Pulse Wave and the Measurement of Arterial Pressure. 351
distended or deformed. Experimenting in the same manner with this
xylol-soaked tube we obtained—
Systolic pressure. | Obliteration pressure.
mm. Hg. mm. Hg.
133 95 (fig. 5)
Thus the rubber tube by soaking in xylol was brought to resemble the
artery.
|
es cl) MH)
4 /
Ud, YY
C spring
733
“Ay yt ol
rubber Pressure tn
\ small bag
Zero
Fig. 5.
Further experiments we have done on human arteries are these :—
I. We bandage the arm with a rubber bandage, place the armlet on the
upper arm, raise the pressure in it above systolic pressure, and then
remove the bandage—the arm is left exsanguined. We now place the
sphygmograph (using the weight extension method) in position on the
radial artery, then let go the armlet. We find that the pulse curve returns
slowly to its full amplitude when the weight extension is 300 grm., while it
returns almost instantaneously when the weight is 150 grm. (see fig. 6,
2 and 3). When the heavier weight is used the pulse wave does not lift it
until the tissues fill with blood and the peripheral resistance increases
to such a degree that the systolic pressure in the surrounding tissues and
artery overcomes the pressure of the sphygmograph pad which is pressing
352 Messrs. Hill, McQueen, and Flack. Conduction of the
upon them. Ifthe arm be not exsanguined the pulse returns at once to its
full amplitude in spite of the heavier extension weight (fig. 6, 1).
Ce con mee me a “a en Ve oY Ye VY
Radial
ED SE EY) ey (SS
Brachial Brachial
Weight o
M (D) Fadialeaee ae
Armlet
released
Radial | Light weight on
radial
M (2) (150 gms.)
Armlet
released
Radial | ;
Heavy weight on
M (3) radial
(300 gms.)
Fig. 6.
II. We surround the upper arm with ice, while the lower arm is immersed
in hot water. After a few minutes the obliteration pressure is taken with
the armlet on the flushed forearm, while the upper arm is still encased in ice.
Under these circumstances we have generally found that the pressure in the
lower arm is 15-20 mm. Hg higher than it was before the experiment.
The ice is then removed from the upper arm and the pressure quickly taken
with the armlet there. In this case the pressure is generally slightly lower
ciate eee Se Armlet round flushed Armlet round upper
PP forearm, upper arm cold. arm, ice just removed.
arm or forearm.
|
mm. Hg. mm. Hg. mm. Hg.
G.s 97 | 120 95
G. S. 97-100 120 95
J. McQ. 130 142 | 125
M. F. 115 125 | 112
M.F 105 105 85-88
— 95 90-95
(Other arm, 105) (15 minutes later)
8. E. 105-108 115 103-105
— 97
(5 minutes later)
105
After application of ice
to forearm
Pulse Wave and the Measurement of Arterial Pressure. 353
(3-5 mm. Hg) than at the beginning of the experiment. In one case, when
the application of the ice had been so long that the skin of the arm had become
red in patches, the pressure in the upper arm was lowered 20 mm. Hg.
It is a remarkable fact that the readings obtained from the cold upper
arm should be lower than those obtained from the flushed forearm.
The tentative explanation we offer of these results is as follows. The
vessels in the tissues of the upper arm under the influence of the ice are
constricted and largely exsanguined, thus the artery is less well supported
by the resonance of the pulse in these tissues, and is therefore deformed by a
lower pressure than is required in the flushed forearm, where the resonance
of the systolic wave is greater. At the same time the cold contracted
artery in the forearm conducts the crest of the wave better to the flushed
forearm since it is less labile. The readings obtained from the cold upper
arm show that the wall of the artery, even though contracted by cold, does
not offer such a resistance to compression as to influence the readings. We
(L. H. and M. F.) reached the same conclusion by methods we devised for
testing the readings we obtained in cases of high blood-pressure.*
Conclusions.
We conclude that the armlet or Leonard Hill’s small bag, applied to the
radial artery, give, under ordinary conditions, accurate readings of systolic
pressure, the obliteration of the pulse being taken as the index. This is
because the artery is surrounded by pulsing tissues and cannot be deformed
until the systolic pressure is overcome in these tissues. The artery is equally
compressed on all sides by a pulsating fluid pressure and the conditions are
the same as when it is compressed in a glass T filled with Ringer’s fluid. In
the dorsalis pedis, the temporal, or aberrant radial artery, where lying on
bone and tendon, the pulse is obliterated by a pressure of the small bag much
lower than the systolic pressure. This is because the lumen of the labile
arterial wall is deformed more easily under these conditions from the circular
to an elliptical shape, and the resistance to the passage of the pulse wave
thereby increased.
The higher the diastolic pressure the greater must be the pressure of the
bag to produce the required deformation. As the amplitude of the pulse
wave depends so much on the size of the lumen, it seems probable that the
higher readings obtained in cases of aortic regurgitation are due in part to the
lumen of the aorta, iliac and femoral arteries being relatively wider than that
of the subclavian and brachial arteries. The pulsating (resonating) support
given to the former arteries by the relatively massive abdominal organs and
* ‘Brit. Med. Journ.,’ January 30, 1909.
354 Floral Mechanism of Welwitschia mirabilis, Hooker.
the tissues of the thigh may also help to prevent the damping of the crest of
the pulse waves. The leg arteries are probably held in a more supported
state, less labile, and for this reason also the pulse will be conducted to the
leg with less diminution in force. Size of lumen, resonance and lability are
three factors which may all take a part in the production of this phenomenon.
We have brought forward in this paper experiments which demonstrate these
factors at work.
This research was carried out with the aid of a grant from the Royal
Society Government Grant.
On the Floral Mechanism of Welwitschia mirabilis, Hooker.
By ArtHuR Harry Cuurcu, Lecturer in Botany, University of Oxford.
(Communicated by Prof. A. C. Seward, F.R.S. Received December 23, 1913,—
Read February 5, 1914.)
(Abstract. )
1. In the preparation of sectional schemes for the flowers of Welwitschia
mirabilis, in different stages of development, several points of interest were
noted as tending to throw light on the previous history of this unique floral
form.
2. Evidence is adduced to show that the primary structural features of the
flowers are referable to an anthostrobiloid condition closely comparable with
that of Cycadeoidea, now expressed in a phase of minimum reduction, and to
be regarded as an example of heterophyletic convergence to a simple floral
construction in the gymnospermic condition.
3. Secondary features of biological interest are mainly consequences of
xerophytic specialisation in the inflorescence; condensation of the whole
system to a “cone” necessitates the extreme flattening of the flower in the
transverse plane, which has led to confusion in the interpretation of the facts
of development; the andreecium is represented by a true whorl of six
members.
4, Similarly, secondary clisanthy in the cone mechanism necessitates special
features in the individual flowers, and accounts for the long exserted micro-
pylar tube of the ovulate flower, and the proriasion mechanism of the staminal
tube in the sterile flower.
Regeneration in Gunda ulvee. 355
_ 5. The working mechanism of the latter is clearly indicated by remarkable
phenomena of the storage and subsequent disappearance of starch in the
andrecial region ; while similar phenomena of starch storage and depletion
in the gyneecial region illustrate the progression from a simple “ drop-
mechanism” to a copious exudation of sugar and the adaptation of the
structure to entomophily.
6. The nectary region of the ovule is retained by the gyncecium of the
sterile flower for the same function, and vascular bundles supplying fluid for
this purpose are not necessarily vestigial. Entomophily is thus probably
antecedent to dicliny.
The Influence of the Position of the Cut upon Regeneration in
Gunda ulve.
By Dorotuy Jorpan Luoyp, B.Sc., Bathurst Student of Newnham College,
Cambridge.
(Communicated by J. Stanley Gardiner, F.R.S. Received January 14,—
Read February 19, 1914.)
I. INTRODUCTION.
In 1899 Hallez (4) made the generalisation that the most important
difference between the regeneration in Triclad and Polyclad Planarians was
to be found in the fact that fragments of the former could regenerate in the
absence of the central nervous system, whilst in the latter some portion of
the cerebral ganglia must be present in order for regeneration to take place.
Child (1) has confirmed the fact that the presence of cerebral ganglia, or at
least intact nerve roots, is necessary for regeneration of the anterior end and
sense organs of Polyclads. The experimental work by the same and other
authors has also established that, among Triclads, the genus Planaria is able
to regenerate completely in the absence of cerebral ganglia. The following
notes, however, show that in another Triclad genus, namely, Gunda, anterior
regeneration is, as in Polyclads, dependent on the presence of the central
nervous system.
The experiments described below were carried out in the Plymouth
Laboratory of the Marine Biological Association during the spring of 1913.
I am greatly indebted to the director and staff of the laboratory for constant
kindness during the course of my work at Plymouth. I also stand under
356 Miss D. J. Lloyd. Influence of the Position of the
obligations to the Royal Society, the Zoological Society, and the University
of Cambridge for the use of their tables at the Plymouth Laboratory.
II. MATERIAL.
The experiments were performed on members of the species Gunda ulve.
G. ulve is a small Triclad Planarian belonging, with other marine planaria,
to the order Maricola, and to the family Gundide (Procerodide). Planaria,
the genus on which most of the work on Triclads has been done, is a fresh-
water form (order Paludicola). |
The structure of G. wlve is quite typical of all Triclads and has been
described by Wendt (8) and Iijima(5). It contains the usual trifid gut, which
opens at the point where the three branches meet into a protrusible pharynx.
The mouth is situated near the hind end, rather more than three-fourths
down the length of the body. The accessory genital organs lie behind the
mouth. The cerebral ganglia are about one-fifth the body length from the
anterior end. Two nerve cords run backwards from the ganglia, joining in
the tail to form a complete ring. The two eyes lie in front of the ganglia,
and at the front of the head are the so-called auricular processes, two in
number.
II]. EXPERIMENTS.
A. Posterior Regeneration.
G. ulve will regenerate tails completely either in the presence or absence
of the cerebral ganglia. Text-tig. I, A, shows the levels at which cuts were
made. Fragments taken for posterior regeneration were head-pieces ADD,
AEE, AFF; middle pieces, BBFF and DDFF. Figs. 1-3 show the progress
of tail regeneration in a worm cut through the middle of the pharynx region
(AFF). For worms cut off in front of the pharynx (AEE, I, 4), it is
essentially similar, except that the newly regulated worm is proportionally
smaller. The figures, which are drawn to scale, show quite clearly that the
new tail is produced by a differentiation of the old tissue.
The next group of text-figures shows the regulation of fragments taken
from the middle of the worm. The characteristic difference in behaviour
between the pieces with and without cerebral ganglia (figs. 5-7 and 8-10
respectively) is quite apparent. The former show regeneration taking place
at both ends of the worm, finally resulting in complete restoration of both
head and tail. The formation of tissue at the head end is seen to check the
rate of growth at the tail end. In fragments without ganglia (figs. 8-10)
no head is regenerated and restoration of the tail proceeds as rapidly as in
the head-pieces (with ganglia) described first. Restoration of posterior parts
Cut upon Regeneration in Gunda ulve. 357
takes place at any aboral surface from DD backwards and is independent of
the presence of the ganglia. Fragment ABB, or fragments equally small
taken from any part of the body, die without any regeneration.
1, Fragment AFF, 14 days; 2. Ditto, 28 days; 3. Ditto, 56 days. 4. Fragment AEE,
32 days. 5. Fragment BBFF, 14 days; 6. Ditto, 28 days; 7. Ditto, 56 days.
8. Fragment DDFF, 14 days; 9. Ditto, 28 days; 10. Ditto, 56 days. A. Diagram
of points of section.
B. Lateral Regeneration.
Animals bisected down the median line (text-fig. II) regenerate completely.
The course of regeneration is shown in figs. 1-3.
--In animals beheaded and then bisected down the middle line (fragment
PD), regeneration occurs along the edge of the lateral wound, but there
is no replacement of the anterior end (text-fig. II, 8-11) unless parts of the
358 Miss D. J. Lloyd. Influence of the Position of the
Li
LO
Fig. II.
1. Fragment AP, 20 days; 2. Ditto, 27 days; 3. Ditto, 41 days. 4. Diagram of points
of section. 5. Fragment CP, 20 days; 6. Ditto, 41 days; 7. Ditto, 62 days.
8. Fragment DP, 20 days; 9. Ditto, 32 days; 10. Ditto, 41 and 62 days. 11. Same,,
side view.
cerebral ganglia are left in the fragment. In this case there is some
regeneration of tissue at the anterior end, but the heads produced are
defective (text-fig. II, 5-7).
Cut upon Regeneration in Gunda ulve. 359
C. Anterior Regeneration.
Anterior regeneration in animals cut anteriorly to the cerebral ganglia is
quite complete (text-fig. III, 1-3). In cases, however, where the cut takes
away more than about one-third of the ganglia, it is only partial. Forms
with either two eyes or one are produced, the auricles in the latter case
being fused (figs. 4 and 5). In animals cut behind the cerebral ganglia
Z Gj
i | ;
40) 6
Fig. ITI.
1. Fragment BBP, 14 days; 2. Ditto, 28 days; 3. Ditto, 41 days. 4. Fragment COP,
41 and 62 days. 5. Fragment CCP, 41 and 62 days. 6. Fragment DDP, 41 and
62 days.
no head formation ever occurs. These posterior pieces showed some change
of form, as forward growth caused them to become helmet shaped (fig. 6).
After two months these pieces still remained without any attempt at regenera-
tion of the head.
A number of headless pieces and defective heads were sectioned for
examination of the nervous system, and the sections are shown.
360 Miss D. J. Lloyd. Influence of the Position of the
It is obvious that in cases of incomplete regulation the central nervous
system is incomplete in corresponding extent.
D. Heteromorphic Forms.
The culture which produced these forms was one of very short head-
pieces where the cut had passed across the anterior part of the brain. The
tail-pieces corresponding to these head-pieces also produced new heads.
The heteromorphic forms 20 days after section are shown in fig. IV. In
cases where the cut passed obliquely the new heads formed on the anterior
end of the cut. This is shown in fig. IV (2).
Z 2
Fie. IV.
land 2. Fragment ACC, 20 days.
A longitudinal section through a heteromorphic form is shown in fig. IX.
The rounded shape of the section is due to the contraction that takes
place when the animals are dropped into the fixing fluid. It can be seen
in the section that about one-third of the complete brain is present. Two
well-marked nerves run to the new eyes, and the portion of regenerated
gut in the new head shows a beginning of the formation of the three branches
characteristic of the front end of the gut of G. ulve. The gut is full ofa
dark brown mass of broken-down tissue, and can be seen clearly in the
whole specimens.
E. Regeneration of Nervous System.
In planarians, z.¢.,in P. torva (Flexner (3) and Schultz (7)) and Planaria
(Dendrocelum) lactea (Schultz (7)) the new nervous system arises by cells
from the parenchyma crowding round the cut ends of the nerve cords and
pushing up among the old nerve fibres, aided possibly by some outgrowth
of the old fibres. This is also the method by which G. ulve repairs injuries
to the nervous system. Thenerve cords of G. ulve always show regeneration
after a cut, and in every case of tail-formation the new nerve fibres can be
found reaching to the end of the newly formed tail at every stage of its
growth, and within a few days of the cut joining at the hind end to form the
posterior commissure.
Cut upon Regeneration in Gunda ulve. 361
After a longitudinal bisection the brain will easily regenerate the removed
half, and the circuit of the nervous system is restored, often before the long
wound has healed over. Transverse sections across the ganglia, however,
are not followed by regeneration of the ganglia if more than about one-third
of the brain is taken away. The nervous system forms a complete ring, and
in every case, where this ring is broken, regeneration of the nerve cords takes
place sufficiently to restore the circuit, though the cerebral ganglia may or
may not be regenerated.
Text-fig. V shows an animal (text-fig. III, 3) where the cut passed across
the front of the ganglia, which were subsequently completely restored. Text-
Fig. VIII (text-fig. III, 6) shows an animal in which the cut removed both
ganglia. In such conditions the nerve cords grow forward and fuse, but no
ganglia are regenerated, and the animals remain headless. Text-figs. VI and
VII show two worms (text-fig. III, 4 and 5 respectively) where the cut has
damaged both ganglia and where there is correspondingly defective restoration
of the head.
Fic. V.—Horizontal Longitudinal Section through Complete Regenerated Head, 41 days
(text-fig. III, 3). ga., ganglion ; n.c., nerve cord ; op.n., optic nerve.
362 Miss D. J. Lloyd. Influence of the Position of the
Fie. VI.—Section through Incomplete Regenerated Head, with two eyes, 62 days (text-
fig. III, 4). emc., excretory canal; ga., ganglion; g., gut; .c., nerve cord; op.n.,
optic nerve.
-€
ao
a IO
BISSS O'S RO --
é “O75
‘Fic. VII.—Section through Incomplete Regenerated Head, with one eye, 64 days (text-
fig. III, 5). ¢., eye; g., gut; ga., ganglion ; op.n., optic nerve.
Cut upon Regeneration in Gunda ulvee. 363
EXC.
pea)
Fie. VIII.—Section through Headless Form, 62 days (text-fig. III, 6). ea.c., excretory
canals; g., gut; 7.c., nerve cords ; pa.c., parenchyma cells ; p.c., pigment cells.
Fic. 1X.—Section through Heteromorphic Head, 20 days (text-fig. IV, 8). ¢,eye;9., gut;
ga., ganglion : op.n., optic nerve ; pa.c., parenchyma cells.
IV. DIscussIon.
As was mentioned earlier in the paper, Child (1) has already shown
that the presence of at least half the cerebral ganglia is necessary for
complete regeneration in Leptoplana. The removal of more than half
VOL. LXXXVII.—B. 25
364 Miss D. J. Lloyd. Influence of the Position of the
the ganglia causes the production of defective heads. When ganglia and
nerve roots are completely removed head-formation is entirely inhibited.
The behaviour of Planaria dorotocephala was found by Child (2) to be
in direct contrast to this. Planaria dorotocephala under normal condi-
tions regenerates the anterior end completely from any point of section.
It is only when regeneration is suppressed by the addition of anesthetics
or potassium cyanide that defective heads are produced essentially similar
to those obtained in Leptoplana and Gunda ulve, 1.e., heads with three to
four eyes, or with a single median eye and with the auricles approxi-
mated or even fused. With increasing defect of the external appearance
Child has shown that there is increasing defect in the regeneration of the
ganglia, eg., In teratomorphic (Child) heads, with single median eye and
fused auricles the cephalic ganglia are partially or completely fused ;
in pieces in which there is no head-regeneration there is similarly no
regeneration of the cephalic ganglia. In fact so closely does the
parallel run that it seems almost justifiable to assume that in Leptoplana,
Planaria, and Gunda head-regeneration is dependent on the presence of the
central nervous system, and that the difference between them is found in
the greater power of regeneration possessed by the central nervous system
of Planaria. Planaria has another characteristic in which it contrasts
strongly with the two other genera, and that is in its power of asexual
reproduction. It seems quite likely that asexual reproduction and the high
capacity for head-formation are both determined by the power of growth and
regeneration of the central nervous system.
In G@. ulve the condition is found that the nerve cords can exhibit restora-
tion by backward and forward growth after a cut made at any part of their
length, quite independently of the ganglia. The ganglia themselves appear
incapable of restoring lost parts, unless one complete ganglion is present, and
the restoration of complete heads only occurs if the ganglia are restored.
Considering the inability of animals with badly damaged central nervous
system to regenerate heads, the production of heteromorphic heads on
short anterior pieces is of great interest. ‘These pieces contain only about
one-third of the cerebral ganglia, yet the heads which they regenerate at
the posterior end are complete with trifid gut, eyes and auricular processes.
It will be remembered that large posterior pieces with defective cerebral
ganglia failed to regenerate heads on the oral pole.
In Planaria maculata Morgan has obtained heteromorphic heads at any
point of section, provided that the fragments which he took were sufficiently
short.
From these considerations it seems possible that the mechanism for the
Cut upon Regeneration in Gunda ulvee. 365
restoration of the tail belongs to the body as a whole, while that for
restoring the head is an entirely independent one, which may or may not
be localised in some part of the body, notably the anterior end. Probably
head-formation is dependent on the presence of some specific substance
which in Leptoplana and Gunda is localised in the anterior end, and in
Planaria is found throughout the length of the body. It would be of great
interest and possibly of great importance if a comparison of the relative
number of ganglia found in the nerve trunks could be made in these three
genera, as it might produce some evidence for or against the suggestion that
the head-forming substances may be localised in the central nervous system.
At present Morgan’s (6) statement that pieces of Planaria maculata entirely
devoid of nervous system are capable of complete regulation rather bears
against the idea that the nervous system is of such great importance in
regeneration. The facts presented in this paper do not justify a full dis-
cussion of Child’s theory of the axial gradient; all that need be said is that
to make this work coincide with his hypothesis the axial gradient of G. ulvw
must be assumed to slope very steeply at the anterior end. Probably the
idea that there is a sudden change in rate of chemical actions in G. ulvw as
one passes backwards from the anterior end is not antithetic to the idea of
the localisation of some specific enzyme in the front of the body.
Finally it may be noted that this work has disproved the suggestion made
by Hallez (4) that Triclads and Polyclads each have their own method of
regeneration, for G. wlve, which is a marine Triclad, behaves like the Polyclads
in its method of the restoration of the head.
Summary.
1. Regeneration of posterior parts is independent of the presence of the
cerebral ganglia.
2. Lateral regeneration behind the level of the ganglia is independent of
their presence. In front of the level of the ganglia at least one complete
ganglion must be present for regulation to occur.
3. Anterior regeneration only occurs if the piece contains about two-thirds
of both ganglia.
4. Heteromorphic heads are formed by short head-pieces where the cut
has passed through the ganglia.
5. G. ulve differs from most other Triclads and corresponds to Polyclads
in its mode of regeneration.
2H 2
366 Mr.8. B. Schryver.: Investigations dealing with the
BIBLIOGRAPHY.
1. Child, C. M., “Studies in Regulation. V and VI.—The Relation between the
Central Nervous System and Regeneration in Leptoplana,” ‘Journ. of Exp.
Zool.,’ 1 (1904).
2, Child, C. M., “Certain Dynamic Factors in Experimental Reproduction and their -
Significance for the Problems of Reproduction and Development,” ‘Arch, f. Ent.
Mech..,’ vol. 35, p. 598 (1913).
3. Flexner, Simon, ‘ Regeneration of the Nervous System of Planaria torva,” ‘Journ.
Morph.,’ vol. 14, p. 387 (1898).
4, Hallez, “Regeneration comparée chez les Polyclades et les Triclades,” ‘Comptes
Rendus,’ vol. 28, sess. 1, p. 270 (1899).
5. lijima, “Untersuchungen tiber den Bau und die Entwicklungsgeschichte der
Siisswasser Dendrocceelen (Tricladen),” ‘ Zeitschr. f. wiss. Zool., vol. 40, p. 359
(1884).
6. Morgan, T. H., “The Control of Heteromorphosis in Planaria maculata,” ‘ Arch. f.
Entw. Mech.,’ vol. 17 (1904).
7. Schultz, E., “Aus dem Gebiete der Regeneration bei Turbellarien,” ‘ Zeitachr. if
wiss. Zool.,’ vol. 72, p. 1 (1902).
8. Wendt, A., “Uber den Bau von Gunda ulve,” ‘Arch. f. Naturgesch.,’ vol. 1, p. 54
(1888).
Investigations dealing with the Phenomena of “ Clot” Formations.
Part IlL—The Formation of a Gel from Cholate Solutions
having Many Properties analogous to those of Cell Membranes.
By S. B. ScHRYVER.
(Comartinien ts! by Prof. V. H. Blackman, F.R.S. Received January 22,Read
February 19, 1914.)
In the first communication under the above title,* attention was called to the
fact that solutions of sodium cholate, on warming in the presence of calcium
salts, set to a gel, which is not reversible on cooling. It has since been found
that calcium salts can be replaced by other salts, such as sodium chloride,
magnesium chloride, ammonium sulphate and potassium fluoride, and the clot
formation is not therefore due to double decomposition between calcium salts
and sodium cholate. The concentrations of sodium, potassium, and magnesium
salts necessary to produce the “clot” are, however, much higher than that of
calcium chloride, which even in the concentration of N/40 can cause 1-per-cent.
sodium cholate to set to a solid gel in about a quarter of an hour at 50°.
Sodium and magnesium chlorides produce clot formation at 50°, when their
* ‘Roy. Soc. Proc.,’ B, vol. 86, p. 460 (1913).
Phenomena of “ Clot” Formations. ; 367
concentration is of about the order of half saturation. It is proposed to inves-
tigate the relative clotting power of salt in greater detail later; the present
communication deals mainly with the question of the influence of one parti-
cular factor on gel formation, viz., on the surface tension of the solution.*
-A preliminary account of the clot production by various calcium salts has
been given in the first paper (Joc. cit.). It was then shown that in the case
of calcium salts which increase the surface tension of water, increase in the
concentration of the salt caused a diminution of the clotting time. In the
ease of the calcium salts which lowered the surface tension, however, the
accelerating effect of the increase in the concentration of the salt was counter-
balanced by the diminished surface tension of the solution. In two cases (those
of calcium dichloracetate and of the sulphocyanide) an optimal point was
found. Increase in the concentration of the calcium salt above and below this
point caused a lengthening of the time required for gel formation. In the
ease of calcium trichloracetate, the length of clotting time progressively
increased with increasing concentration within the limits investigated.t
Method of Experiment.—The following was the method of experiment
adopted :—1 c.c. of a 4-per-cent. sodium cholate solution and 3 c.c. of the salt
solution in the required concentration were heated in separate quartz tubes
of 10 c.c. capacity in a thermostat at 50°. Assoonas the liquids had attained
the temperature of the thermostat, they were rapidly mixed ; the salt solution
was poured into the cholate solution, and the mixture was then poured back
into the tube originally containing the salt alone. This was then clamped if
the (transparent) thermostat and watched. The formation of oily globules was
the first sign of clot formation. These at first moved rapidly in the liquid, but
as they increased in size, motion became less rapid, until, finally, movement
was hardly visible. At this point, at short intervals, steel balls of 3/32 inch
diameter, such as are used for ball-bearings, were dropped into the tube at
short intervals. The time of complete gel formation was taken As that at which
a ball stopped dead before it had fallen half-way through the tube. The time
was taken by a stop-watch, which was started at the moment of mixing the
solutions. Even when the clotting time was 5 minutes, control determinations
seldom differed by more than 10 seconds—they usually agreed with one another
within 5 seconds. In the first series of experiments on calcium salts, the cholate
solution was made by saturating N/100 sodium hydroxide with cholalic acidt
* Throughout this communication by “surface tension” is meant surface tension
measured against air.
+ The surface tension of the salt solutions is affected mainly by the anions. The series
of anions employed was that used in the investigations on aggregation (‘ Roy. Soc. Proc.,’
B, vol. 83, p. 96 (1910) ).
{ Prepared pure by the author’s method (‘ Journ. Physiol.,’ vol. 44, p. 265 (1912) ).
368 Mr.S. B. Schryver. Investigations dealing with the
until a solution neutral to neutral red was obtained. An approximately
4-per-cent. solution was obtained. It was found, however, that cholalic acid
is soluble in sodium cholate solution, the amount dissolving varying with the
temperature. As this free acid inhibits the clotting, it was found that the
clotting time of a solution varied from day to day. For all subsequent
experiments, sodium cholate was prepared and a 4-per-cent. solution was made
directly from this. The cholate was made by dissolving cholalic acid in 20
times its weight of alcohol, neutralising this solution with sodium ethoxide,
heating for a short time on a water-bath and filtering off the first separation
of solid, and then evaporating the filtrate. Sodium cholate rapidly separated
after a short time, and was filtered off, washed with acetone, and then dried,
first on a water-bath, and then over sulphuric acid in a desiccator. It gave a
solution in water, acid to phenolphthalein, but slightly alkaline to neutral red.
Table I—Clotting Time of 1-per-cent. Sodium Cholate Solution (in seconds)
in presence of Varying Concentrations of different Calcium Salts.
| 3N/4. N/2. 3N/8. N/4. .| N/B.
Chiorideyeremeeep eee eaeeee 30 14 14°0 21 34
Bromide i255. \kceceeee cence 10°5 14 15 °5 21 47
NGtrabe ,ge:1800
Phenomena of ‘“ Clot” Formations. 371.
Table [1—continued.
|
|) @rm. © | Clotting | @rm. Clotting Grm. | Clotting
yal). i | mol.per time, | ae 5 ue mol. per time, * Gaby Mp mol. per time,
| i | litre. Secsimaliwi hen?” | litre. secs. Pus litre. secs.
| |
|
|
Ethyl Carbaminate. Propyl Carbaminate. | Witte’s Peptone.
Per cent. | | | |
(weight). | |
1°25 0°140 | 26 Leb) Maral 36 | «1875 213
1°875 0°210 | 38 1°875 0182 93 2°5 360
2°5 0°281 | 56 =| 2°1875 0-212 248 |. 3°125 570
3°75 0-421 | 147 2°65 0-242 360 | 3°75 1530
4.875 0-491 296 3 °125 0-303 910 |
5°0 O°’561 | 564 | Phenol.
5625 | 0-632 | 750 | | |
6°25 0 °702 | 1448 | 0 °625 165
| 0 ‘9375 550
—
Chloroform.
A solution of water saturated with chloroform at 17° contains 0°710 per
cent. When 2:5.¢.c. of this solution was mixed with 0:5 cc. N. calcium
chloride and 1 ¢,c. 4-per-cent. cholate solution, the clottmg time was not
appreciably longer than when no chloroform was present. By diminishing
the concentration of the calcium salt to one half, the clotting time was
230 seconds in the presence of saturated chloroform water (2°75 cc. in 4
= 0°041 grm. mol. per litre), as compared with 45 seconds, the clotting time
in the absence of chloroform. The clotting time in presence of 0°064 grm.
mol. per litre amyl alcohol, and the same amount of calcium salt, was
155 seconds, and of 0:038 grm. mol. per litre chloral hydrate 194 seconds.
The inhibitory action of chloroform is therefore greater than that of amyl
alcohol. :
Nitromethane.
The action of nitromethane is anomalous. In the presence of 3°75 per
cent. the clotting time is 23 seconds, in the presence of 5 per cent. it is
57 seconds, and of 6°25 per cent. it is 42 seconds. It appears to behave more
or less like an acid, for in the presence of hydrochloric acid in N/800 con-
centration, the clotting time of cholate solutions is 41 seconds, in N/400 it
is 104 seconds, 3N/800 it is 185 seconds, and in N/200 it is 74 seconds. At
the highest of these concentrations the acid is sufficient to cause precipitation
of free cholalic acid; on keeping at 50° the precipitate disappears and a gel
then forms. The lower concentrations produce no separation of free organic
acid in form visible to the naked eye. The nitromethane possibly forms the
GY ae
salt CH;N—ONa by double decomposition.
\OH
372 Mr.8. B. Schryver. Investigations dealing with the
Polyhydroxy-Derwatives,
The inhibitory action of these substances is small, as is shown by the
following examples :—
Substance. Per cent. (weight). Clotting time, secs.
Ethylene glycol............ 12°5 281
Propylene) 3." .acaseesece: 8°75 385
(with separation of crystals)
Giycerol@ercen- ce saereeeeeee 12°5 148
Sucrose bs) .ces-eeeeeere cae 12°35 29
iDext Tose pereeery saeereerer eee 12°5 30
Dextriniycs....cocsureesee 12°5 258
Phy My) so nopedeacadeasccsueas 10-0 100
Discussion of Results.
Whilst it cannot be denied that those substances which lower most
markedly the surface tension of water have, as a rule, the greatest tendency
to exert an inhibitory effect on the formation of the cholate gel, the law is
not by any means an absolute one. The exceptions are precisely the ones
which deviate from Czapek’s generalisation. Acetonitrile, which lowers the
surface tension of water but little, has a greater inhibitory power than ethyl
aleohol, which lowers it much more. The deviation from the rule is shown
in a very marked manner also by the typical narcotics, chloral, chloroform,
and (in the experiment on gel formation) by urethane. There is, in fact, a
striking parallelism between inhibition of gel formation, narcotic and
hemolytic actions and production of tannin exosmosis, which is well
exhibited in the following table. The various substances are arranged
Substances in decreasing order of Critical narcotic concentration.
gel-inhibiting action. Grm. mol. per litre.
CHLOROFORM” 7 i5,5. cise cent nace ce eee aes eae ene eee 0 0012
CHLORAGHY DRAPE! (.25.. tessse cence seaceeaceeemeenate eee ee seeeert - 0°02
Tsoamyl] alcohol.c:tsycaaecesasiGies ec ee eee ee ee 0-023
Secondary amyl alcohol (methyl propyl carbinol) ......... =
Tertiary amyl] alcohol (dimethyl ethyl carbinol)............ 0 :037
Propylicarbaminatel--sne-saceeenesrce eer eert eee ceeeerer emer eee =
Noxnral foutylvallcoholieescsessa scence tenet eee ates 0 ‘038
(lethylipropyliketone) -ereasseceeee cena eee eee eee 0-019)
Tsobutyaleohol yc cssccuees ae ee eee ee ana eS EERE 0°045
Normal jpropyliialcoholey ye ee eee ee eee cee eae eee O-1l
UR RDRVANIE (505, Set. scm acca taeses dann pesoeeeeaiee deteatee meee ace 0 ‘041
MertiaryAbuby lial cohol ce eeereeeee eee eereeaaeene ee eee cee 0-13
Tsopropyl@leohol! .30.c5-caause-seeeeceeseee eee eee nee 0°13
AM YL alcohol sca sc.\ 0s sect oer ee ook cee Ree eee ee 0°18
Methyl jcarbaminaitesss..roecesepepeeeeeserees see ne eaceeet ren: nee 0°27
ACETONUDRIGE) 5.608 sie shire decth sane a eeeelenaeee asec eEoaee 0-36
Ethylialcoholl v.53) anetinnas ses bcecsecoasns reek ae meee eeeeee 0°3
Phenomena of ‘‘ Clot” Formations. 373
in the order in which they inhibit the gel formation, the more active
substances being placed first in the list. The numbers given are the
strengths in which they produce narcosis of tadpoles according to Overton,
The concordance between the gel inhibitory action and the narcotic action
is striking. Methyl propyl ketone is an apparent exception, but gel-inhibiting
action of this substance cannot be accurately determined, as in relatively
small concentrations it causes the formation of crystals. The same is true
for ethyl aleohol in higher concentrations. Normal propyl alcohol should
follow instead of preceding urethane. The substances showing a marked
deviation from the surface tension generalisation are indicated in large
type.*
General Summary and Conclusions.
The inhibition of gel formation may be assumed to be due to adsorption of
various substances from solution, which prevent the formation of larger
ageregates, which constitute the gel.t The adsorbability of those substances
cannot be determined by their effect alone in lowering the surface tension of
water. Czapek has assumed that certain plant cells have a lipoid membrane,
with a surface tension of about 0°681 (water = 1), and that, when they are
immersed in an aqueous solution, the surface tension of which has been
reduced to below this figure, exosmosis of complex molecules takes place,
owing to the changes in the lhpoid membrane. Czapek found, however, that
certain substances deviated from his rule. To these he ascribed a specific
toxic action on the cell. In view of the fact that these same substances
show a deviation also from a surface tension rule in their inhibitory action
on the formation of the cholate gel, a phenomenon from which specific
biological action is excluded, the purely mechanical conception of cytolysis,
as propounded by Czapek, is clearly no longer tenable. Nor do the results
in the above paper support the Overton-Meyer lipoid hypothesis. Although
the lipoid soluble substances have, as a rule, the greatest inhibitory action on
gel formation, the gel itself cannot, by any extension of the meaning of the
term, be described as a lipoid. It is formed from the salt of an acid, which
is generally insoluble in organic solvents, in which even the free acid itself
is only slightly soluble. The results suggest that the semipermeability of the
cell inay owe its properties to the presence of some gel-forming substance
* Several estimations of the surface tensions of solutions have been made by different
observers. Czapek’s own numbers have been adopted. In arranging the above table the
approximate dilutions which delay gel formation 15 minutes have been ascertained. The
surface tensions of these dilutions in water lies normally between 0°5 and 0°67 (water = 1).
The substances indicated in capitals deviated markedly from these numbers.
+ Compare Schryver, ‘ Roy. Soc. Proc.,’ B, vol. 83, p. 96 (1910).
374 Investigations dealing with Phenomena of “ Clot” Formations.
which has not yet been isolated, and which need be neither lipoid nor
protein. Such a gel need not, furthermore, be continuous, but may simply
form a matrix, holding together proteins and lipoids and other cell con-
stituents. The protoplasm itself may exert its normal functions only when
its constituents are held in such a matrix. The amount of substance to
which the gel formation may be due need be present only in very small
quantities. A solid gel has been obtained with 4-per-cent. solutions of
sodium cholate, but the author, in conjunction with Dr. E. Graf von
Schénborn (in a preliminary communication to the Biochemical Society
last May), has shown that solid gels are formed from sodium deoxycholeate
(another bile acid), when the concentration does not exceed 1 in 1000.
Various other problems arise from the study of these gels. Attention has
been called to the fact that relatively large quantities of sodium and
magnesium salts are necessary to produce gel formation as compared with —
those of calcium salts. These facts offer a suggestion as. to the antagonism of
calcium salts to the toxic action of sodium and magnesium salts, as has
been observed by Loeb, in the case of fundulus eggs, and of which many
other biological examples exist. The replacement of a calcium salt by
sodium or magnesium salts may render a gel unstable. It is proposed to
investigate phenomena of this description. In all the above experiments
a large excess of calcium salts has been employed in gel formation, in order
to accelerate this phenomenon. To obtain results more analogous to the
various biological phenomena, it will be necessary to study the action of
various reagents on the gel when in thin membranes, and under conditions
under which excess of calcium salts can be readily removed.* Preliminary
experiments indicate that under such conditions the gel may be reversed.
Work is proceeding in this direction, and it is also proposed to employ the
gels for the study of various phenomena of permeability.
* In the above-described experiments the inhibitory action of various substances on a
membrane (or gel) formation has been studied. It has been assumed in these arguments
that the more powerful this particular action of a given substance is, the greater will be
its disaggregating action on an already formed membrane (or gel).
_ 3875
A New Malaria Parasite of Man.
By J. W. W. StepueEns, M.D., Sir Alfred Jones Professor of Tropical
Medicine, University of Liverpool.
(Communicated by Sir R. Ross, K.C.B., F.R.S. Received January 19,—
Read February 19, 1914.)
[Puates 14-16.]
In the autumn of 1913 Major Kenrick, I.M.S., kindly sent me, from
Pachmari, Central Provinces, India, a blood slide from a native child,
containing numerous malaria parasites. On examining these, which I at
first took to be malignant tertian parasites, the suspicion arose in my mind
that there was something peculiar about their appearance. I happened just
previously to have been studying a blood slide from Rhodesia, containing
very numerous malignant tertian parasites. The peculiarity of the Indian
parasite, as far as I could at first define it, was that it was an irregular
parasite as compared with the regular, almost monotonous, contour of the
“rines” of the malignant tertian parasite (Plasmodium falciparum). I
proceeded then to study the Indian parasite more carefully; and, after daily
observations for many weeks of it, and of control malignant tertian
parasites from various sources, I came definitely to the conclusion that it
was unlike any malignant tertian parasite that I had ever seen or that I
could find figured in the text-books or journals. I also considered carefully
the possibility of its being the simple tertian parasite, but to this point I
shall return later. During this study, in order to fix my impressions, I drew
- 150 consecutive parasites from the Indian slide and the Rhodesian slide
respectively, as the former appeared in the field of view of an ocular so
restricted by placing a diaphragm in it that only half a dozen red cells were
visible in the field at a time, thus effectively preventing any selection on
my part. I reproduce as pen-and-ink drawings 35 of each series taken at
random, as they show very well in a general way the different aspect of
the two parasites, For the same purpose I also drew a number of young
simple tertian parasites.
I now proceed to define as far as possible in detail the peculiarities of
this parasite,
1. It is extremely ameboid (judging from the stained specimens), Thin
processes often extend across the cell or occur as long tails to more or less
ring-shaped bodies. These processes may be several in number, and may
376 Prof. J. W. W. Stephens.
- give the parasite most peculiar fantastic shapes like that of an irreeular
web or mesh.
2. The cytoplasm is always scanty, 7.e. the individual ameeboid processes
are delicate or thin, and the parasite has but little bulk, or density. While
forms resembling “rings” do occur, yet, owing to the abundance of all kinds
of irregular forms, it is certainly difficult to find quite typical “signet”
rings. Laterally applied parasites (accolés of French authors) also occur, but
in them the chromatin is not dot-like, as it usually is in the malignant tertian,
but practically always rod-like.
3. The nuclear chromatin is out of proportion to the volume of the
parasite. It takes the form of bars or rods, strands, curves, forks,
patches, etc.; the occurrence of the chromatin in a dot, as in the “ring”
forms of other species, is rare. In the web-like protoplasmic processes
mentioned above there may be seen several chromatin strands, and not
uncommonly one observes a minute dot of chromatin some way from
the parasite, or between two portions of the parasite, though the
protoplasmic process connecting it with the main mass or masses is so thin
as to be invisible. The chromatin masses are frequently angular, the angles
jutting into the points at which an ameeboid process is given off. Abundance
of, and marked irregularity in distribution of, the chromatin masses are
characteristic of this parasite.
I reproduce in a coloured plate the peculiar forms of this parasite, as it
is very difficult, if not impossible, to describe them in words.
I next consider in what respects this parasite in my opinion differs from
the hitherto described parasites of malaria.
Malignant Tertian Parasite—It differs from this
(1) In its ameeboid activity. In the case of the malignant tertian parasite
a certain amount of amceboid activity is observable, giving rise to “ star-fish”
shapes, and to somewhat irregular or even bacillary forms; but the activity
is not comparable with that of this parasite, which has for this reason a most
strange and peculiar appearance. The picture produced by the splash of a
drop of ink on paper may suggest some of the forms seen.
(2) In the abundance and irregularity of nuclear matter. This, as the
coloured plate shows, is very different from what one finds in the malignant
tertian parasite, where the term “signet rings” well expresses the general
morphology. The quotidian parasite, if such exists, differs so slightly
morphologically from the malignant tertian parasite that the differences
just pointed out between this Indian parasite and the malignant tertian
apply equally to it.
Simple Tertian Parasite —It differs from this in the following respects :—
Sltepheses. Roy. 0c. Proc. B, vol.34 PL14.
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A New Malaria Parasite of Man. 377
(1) Its bulk is much less, 2c. it is a smaller parasite.
(2) The ameboid processes are far more delicate.
(3) The chromatin shows a relative abundance, an irregularity and a
peculiarity of arrangement (eg. strands, rods and bars) not seen in the
simple tertian parasite.
(4) Typical rings are absent or exceedingly rare.
I am not sure whether this parasite enlarges the cell, as, although in some
eases I found infected cells larger than non-infected ones in their vicinity,
in other cases the reverse held good. I am uncertain also whether it is
pigmented or not. I have found no parasites in which I could certainly
detect pigment; but, on the other hand, I found three pigmented leucocytes
in the film, which leucocytes may be associated with this parasite or may
result from an associated infection. Finally, 1 am in doubt as to whether
it produces any change in the red cell such as Schiifiner’s dots. During
the course of my examination of this film 1 must have observed many
thousand parasites, but among these I encountered only one infected cell
which was clearly enlarged and which showed Schiiffner’s dots. The bulk
of this parasite was much greater than that of any other I had seen,
whereas the chromatin masses (two in number, one large, one small) were
small compared to the bulk of the parasite. Although I could detect no
pigment in this parasite I was not otherwise able to distinguish it from a
simple tertian parasite.* These points then must remain unsettled until
further material is forthcoming.
Quartan—lIts amoeboid activity and its tenuity easily distinguish it from
this species.
After a prolonged study of this parasite I believe then that its morphology
differentiates it from any malaria parasite of man yet described.
I propose to call it Plasmodium tenue.
DESCRIPTION OF PLATES.
The figures were all drawn with an Abbé camera lucida at the same magnification,
x 2300 (approx.).
Plate 14.—Plasmodium falciparum. Blood slide from Rhodesia ; 35 parasites drawn at
random.
Plate 15.—Plasmodium tenue. Blood slide from Central Provinces, India; 35 parasites
drawn at random.
Plate 16.—Plasmodiwm tenue. Illustrating the irregularity of form of this parasite.
* J incline to the view, however, that this large form belongs to the other irregular
forms, and hence that this parasite-enlarges the cell and produces a stippling in it,
and hence also that it has affinities with the simple tertian parasite, and Plasmodium
canes of the dog rather than with the malignant tertian parasite.
378
Formaldehyde as an Oxidation Product of Chlorophyll Extracts.
By CHARLES HORNE WARNER, B.Sc., F.I.C.
(Communicated by Prof. V. H. Blackman, F.R.S. Received February 3,—
Read March 5, 1914.)
(From the Department of Plant Physiology and Pathology, Imperial College of Science
and Technology.)
Of recent years the action on carbon dioxide of chlorophyll in witro has
assumed some importance as possibly throwing light on the nature of the
photo-synthetic process of green plants.. Thus Usher and Priestley* have
stated that films of extracted chlorophyll in the presence of moist air and
carbon dioxide produce formaldehyde and hydrogen peroxide under the
influence of light. The earlier work: of these authors has been adversely
commented upon by several writers, notably by Ewart,t to whose criticisms
Usher and Priestley have replied with a number of additional experiments
and arguments, referring also to the work of Schryver,t subsequent to that
of Ewart, as affording strong confirmation of their views as far as the
synthesis of the aldehyde is concerned. The facts set forth in the present
paper came to light during an attempt to confirm and extend the observa-
tions of Usher and Priestley and of Schryver.
Grass was extracted with alcohol, usually in the cold and in the presence
of calcium carbonate. In some experiments the alcoholic liquid was
evaporated to dryness under reduced pressure and the residue extracted
with ether; in others a solution of chlorophyll in light petroleum was
obtained by shaking the alcoholic solution with that liquid. The method of
experiment was based upon that described by Schryver, the ether or
petroleum extract being allowed to evaporate on glass plates and exposed to
light under the various conditions to be mentioned below. As was the case
in the later experiments of Usher and Priestley themselves, the test which
has been relied upon for the detection of formaldehyde is the very delicate
one devised by Schryver, who has found that the reaction is not given by
such other members of the series as have been examined up to the present.
It has been assumed in the course of these experiments, as in the work
of the investigators already mentioned, that the aldehyde produced is
* ‘Roy. Soc. Proc.,’ B, vol. 77, p. 369 (1906) ; vol. 78, p. 318 (1906) ; and vol. 84, p. 101
(1911).
+ Ibid., vol. 80, p. 30 (1908).
t Lbed., vol. 82, p. 226 (1910).
Formaldehyde an Oxidation Product of Chlorophyll Extracts. 379
formaldehyde, but it is very important that the possibility of the observed
effects being due to some other aldehyde or to a mixture of aldehydes should
be borne in mind, This point still requires investigation.
The Production of Formaldehyde by Chlorophyll Extracts in Arr.
In 14 experiments films of chlorophyll extract together with tubes of soda
water were exposed to light in glass jars with well-fitting stoppers greased
with vaseline. The effect both of electric light (eight experiments) and
sunlight (six experiments) was investigated. In the former case the source
consisted usually of one or two 32 cp. filament lamps which were separated
from the vessels containing the films by a glass tank, 2 inches thick,
through which cold water flowed; the exposures varied from 6 to 70 hours.*
The films illuminated by sunlight were exposed outside a south window for
periods ranging from two and a quarter hours of bright sunshine to seven
days bright at intervals (March 4-11, 1913).
Similar films were always exposed in jars containing tubes of potassium
hydroxide solution. These control films were allowed to stand in the dark
im vacuo over lime for several days before being rapidly placed in the jars,
and then for several more days over the potash solution before exposure to
light. In all the films a development of formaldehyde was observed, the
solutions becoming very decidedly coloured in most instances when the test
was applied. In 10 of these experiments no difference could be detected
between the amounts of aldehyde formed in the presence of carbon dioxide
and in its absence. In three there was a very slight excess in the films
exposed to carbon dioxide as compared with the control films, but the
difference was so small as to be barely perceptible, while in the remaining
experiment slightly more aldehyde was found in the film which had not
been exposed to carbon dioxide.
Subsequently three experiments were carried out with somewhat greater
precautions, sealed glass tubes replacing the stoppered jars throughout.
The exposures varied from one to two and a half hours of bright summer
sunshine, and in each case the production of formaldehyde was very evident.
In one instance it was impossible to distinguish between the amounts
formed in the presence and absence of carbon dioxide, while in the other
two there was slightly more formaldehyde produced in the films which had
been exposed over potash solution. In all of the 17 experiments control
.films were also examined which had remained in the dark for equal periods,
* During the daytime these films received diffuse sunlight in addition to artificial
light.
VOL. LXXXVII.—B. 2k
380 Mr. C. H. Warner. Formaldehyde as an
and in no case was formaldehyde found in such a film whether carbon
dioxide had been present or not.
In some, at least, of the experiments which yielded indications of the
photo-synthesis of formaldehyde to previous investigators, either lime, soda-
lime, or solid potash was used to obtain a control atmosphere free from
carbon dioxide. A number of additional experiments have been carried out
under these conditions, which are, however, entirely unsatisfactory, sinee the
effect of a moist atmosphere containing carbon dioxide is compared with that
of dry carbon-dioxide-free air. In a few of these experiments, particularly
when short exposures to rather dull light were given, distinctly more
formaldehyde was formed in the films exposed to carbon dioxide tham m the
controls; in the remainder no appreciable difference was observed.
In order to further investigate the influence of moisture upon the
production of formaldehyde, several experiments were performed in such a
way that the effects of moist and dry carbon-dioxide-free air could be
compared. Films were exposed in pairs, one in air over potash solution, and
the other in air over lime or soda-lime, a tube of concentrated sulphuric acid
being present in addition in some eases. In this series also it was noticed in
some instances that the comparatively dry films contained distinetly less
formaldehyde than the moist ones. These experiments, taken as a whole,
appear to show that, while there is no appreciable difference between the
amounts produced by such chlorophyll films in moist air containing carbon
dioxide and in similar air free from that gas (such differences as have been
observed being chiefly due, as shown by later experiments, to the air diluted
by carbon dioxide causing less oxidation), the aldehyde is formed more
readily in moist than in dry air.
It is evident that formaldehyde was being produced in these experiments
as the result of photo-chemical decomposition of the films. This is one of the
points to which attention was drawn by Ewart (Joc. cit.), who found that an
aldehyde was formed by chlorophyll under the action of hght both in air in
which carbon dioxide was present and in that from which it was absent, nor
could he observe any difference in colour intensity between the two cases
when Schiff’s reaction was applied. In this connection Ewart relied chiefly
upon observations on killed leaves, a method of experimentation very
difficult to control; in addition to chlorophyll, the number of substances
present, some of which (such as organic acids) may give formaldehyde on
exposure to light, is very large, much greater than in an ether or petroleum
extract ; also, it is considerably more difficult to ensure freedom from carbon
dioxide for control experiments in such leaf material than in the thin films
obtained in the manner described. Again, the rosaniline test, apart from its
Oxidation Product of Chlorophyll Extracts. 381
comparative lack of sensitiveness and the fact that it cannot be used
quantitatively, must be regarded as most unsatisfactory for work of this
type even when applied with great care, since it has no specificity, and the
escape of sulphur dioxide from the solution causes the appearance of the red
colour in the absence of aldehyde.
Illumination of Films in an Atmosphere of Nitrogen.
3p) J
It was next necessary to determine the nature of the decomposition
described above, in order to eliminate it, and thus ascertain whether photo-
synthesis was taking place at the same time. With this object in view,
films in sealed glass tubes containing alkaline pyrogallate solution were
exposed to bright sunlight for periods of one, one and a half, two, and
two and a quarter hours, and to sunshine intermittently bright for periods of
five and a half and twelve hours (twice). No trace of formaldehyde could be
detected in any of these films after exposure. In carbon-dioxide-free films
which received equal exposures over potash solution, the amounts formed
were always very considerable. .
A tube containing recently boiled water and a film of chlorophyll extract
was six times alternately exhausted and filled with nitrogen, which had
been passed through potash solution and over copper heated to redness.
After the final fillmg the tube was sealed off and exposed to bright
sunlight for two hours, and to diffuse sunlight for a further period of three
hours. On examining the film, again no trace of formaldehyde could be
found, although the quantity which had been produced in a film similarly
exposed over a solution of potassium hydroxide was most distinct. Both
films were allowed to stand 7m vacuo over soda-lime and pyrogallate solution
in the dark for three days before being quickly introduced into the tubes,
and the sealed vessels remained for four days more in the dark before
exposure.
Illumination of Films in Carbon Dioxide free from Oxygen.
Four tubes containing freshly boiled water and chlorophyll extract films
were six times alternately exhausted and filled with carbon dioxide freed
from oxygen by means of red hot copper, and sealed off after the sixth
fillg. The following exposures were given: (#) 12 hours of sunlight,
bright at intervals; (6) 11 hours of bright and 6 hours of diffuse sunlight ;
(c) 25 hours of bright and 6 hours of diffuse sunlight; and (d) 17 days of
intermittent sunshine (July 16 to August 2, 1913). In no case was there
any evidence that formaldehyde had been formed in the exposed films,
although in each case control films, carefully freed from carbon dioxide and
2F 2
382 Mr. C. H. Warner. Formaldehyde as an
exposed over potash solution under the same conditions, showed a very
marked formation of the aldehyde.
Usher and Priestley* thermo-electrically determined the temperatures of
two similar films of chlorophyll extract exposed to light, carbon dioxide being
present in the surrounding atmosphere in one case and absent in the other.
They found that the temperature of the film in contact with carbon dioxide
was the lower, a result which they regard as evidence that synthesis had
taken place consequent on the absorption of energy by the film. This result
is in no way conclusive, and may be quite valueless, since, apart from the
difficulties due to the conditions of the experiment, there is the fact that
oxidation must have been proceeding in both cases, and the observed tem-
perature difference was in all probability due to the excess of oxidation of
the film in air as compared with that of the one in air diluted with carbon
dioxide. The greater proportion of oxygen in the air from which carbon
dioxide was absent would certainly account for the observation that “the film
in CO2-free air was. scorched and destroyed sooner than the other.” The
experiment should be repeated with nitrogen as an atmosphere for the
control film.
The Bleaching of Chlorophyll.
Chlorophyll in air becomes bleached by light both in the presence and
absence of carbon dioxide, and it has been found that formaldehyde has been
produced whenever bleaching has occurred. The bleaching was not more
marked in moist air containing carbon dioxide than in air standing over a
solution of potassium hydroxide, but when the effect of air containing appre-
ciable quantities of water vapour was compared with that of an atmosphere
relatively dry, it was found that the degree of decolorisation (especially in
the case of rather short exposures to comparatively dull light) was somewhat
greater under the moist conditions. In all ‘of the experiments previously
mentioned in which films were exposed in tubes containing no oxygen,
including the four for which oxygen-free carbon dioxide was used, no bleach-
ing could be detected. It will be observed that these results agree with those
which were described when the production of formaldehyde was under
consideration.
The Formation of Hydrogen Peroaide.
The observations of Usher and Priestley on the action of sheep’s liver
catalase in preventing the bleaching of chlorophyll have been confirmed.
These observations point to the conclusion that the bleaching is due to the
* © Roy. Soc. Proc.,’ B, vol. 84, p. 107 (1911).
Oxidation Product of Chlorophyll Extracts. 383
oxidising action of hydrogen peroxide. Less important evidence tending in
the same direction has been obtained by treating films which have been
exposed to light and air with potassium iodide, ferrous sulphate and acetic
acid. A small amount of iodine was slowly liberated, while films which had
been. kept in the dark when treated with the same reagents caused no
liberation in an equal time. A film which had been illuminated in a sealed
tube containing moist oxygen-free carbon dioxide was not bleached and did
not give the above reaction, although iodine was set free by a film similarly
exposed in air over potash solution; this film was very distinctly bleached.
It is, of course, not to be expected that such an unstable substance as hydro-
gen peroxide would accumulate in any quantity in these films.
There thus seems to be good ground for believing that the bleaching of
chlorophyll in oxygen is due partially, if not entirely, to the action of hydro-
gen peroxide. Since bleaching does not occur in oxygen-free carbon dioxide,
however, there is no evidence that carbon dioxide plays any part in the
formation of the peroxide, which substance is obviously produced through the
agency of atmospheric oxygen. The somewhat greater readiness with which
bleaching takes place in moist air as compared with air which is relatively
dry, is doubtless related to these facts, although it would seem that the
change can be effected in the presence of only a very small amount of water.
Usher and Priestley* found that chlorophyll films in a carbon-dioxide-
containing atmosphere which previously produced no effect on Beyerinck’s
“luminous” bacteria, caused glowing after exposure to light. This does not
by any means necessarily point, as these authors consider, to the decomposition
of carbon dioxide with the formation of oxygen or hydrogen peroxide. It is
more probable that, although the gas did not originally contain sufficient
oxygen to cause visible luminosity of the bacteria, sufficient was present to
form hydrogen peroxide under the action of light, and the peroxide, according
to Usher and Priestley, has more effect upon these organisms than oxygen.t
The same criticism is applicable to Molisch’st statement that assimilation can
be carried out by chloroplasts from dried and apparently lifeless cells, and
probably to other observations depending upon the use of bacteria also.
Since the production of formaldehyde always accompanies the disappearance
of the green colour of chlorophyll in air, it is probable that in this bleaching
* © Roy. Soc. Proc.,’ B, vol. 84, p. 105 (1911).
+ Usher and Priestley found that a certain amount of glowing was produced in the
presence of catalase ; hence it may be argued that the effect is not due to the action of
hydrogen peroxide formed in the manner here suggested. It is practically impossible,
however, that under the conditions of the experiment (g.v.) the catalase could render
the peroxide entirely ineffective.
t ‘ Bot. Zeit.,’ vol. 62, p. 1 (1904).
384 Mr. C. H. Warner. Formaldehyde as an
process hydrogen peroxide oxidises chlorophyll with the formation of
formaldehyde among other colourless products, and in support of this view it
may be mentioned that the aldehyde is formed in the dark when films of
chlorophyll extract are immersed in hydrogen peroxide solution.
Whether these transformations play any important part in the metabolism
of the plant remains to be seen. Possibly some group in the chlorophyll
molecule suffers decomposition, thereby liberating formaldehyde, and is then
regenerated under the action of carbon dioxide, but the occurrence in the
tissues of catalases, which may partially or wholly prevent any Shee oxidation
in the living leaf, must be borne in mind.
The observations of Bach and Chodat,* which appear to have escaped
attention in this relation, to the effect that plants (in their experiments,
fungi) are able to live in a medium containing 0°68 per cent. of hydrogen
peroxide, may be found to have an important bearing upon the subject under
discussion.
Experiments testing the possibility of the photo-synthesis of formaldehyde
by colloidal chlorophyll in the presence of plant catalases and other enzymes,
z.e. under conditions more nearly approaching those of assimilating tissues,
might yield interesting results.
Films of Carotin Extract.
A few preliminary experiments have been carried out with films of
carotin extract. Carrots were extracted with hot alcohol and the liquid was
shaken with light petroleum, the petroleum solution being then allowed to
evaporate on glass plates. When such films are exposed to air they become
bleached both in the light and in the dark,t and formaldehyde is produced in
both cases. A carotin film exposed to light in a sealed tube containing moist
carbon dioxide free from oxygen was not bleached and showed no evidence
of formaldehyde development, while a similar film exposed over potash solution
beside the first for the same time became completely bleached and gave a
decided reaction for the aldehyde.
Experiments are now being arranged by means of which it is hoped that
the formation of hydrogen peroxide by chlorophyll and possibly by carotin
and other similar substances, and their derivatives, may be more or less
quantitatively investigated.
Thus far the experiments have been confined to crude chlorophyll- and
* ‘Biochem. Centralblat.,’ vol. 1, p. 417 (1908).
+ Compare Willstitter and Escher, ‘Zeit. Phys. Chem.,’ vol. 64, p. 47 (1910).
Oxidation Product of Chlorophyll Extracts. 385
carotin-containing extracts prepared as described; it is intended shortly to
investigate similarly the behaviour in this relation of the isolated leaf pigments
and some of their derivatives. This is especially important in view of the very
recent work of Spoehr,* who has shown that various acids which occur in the
leaves of succulent plants may be decomposed by light, yielding formaldehyde.
A decomposition such as this would well account for the formaldehyde which
Kimpfiin} found in the leaves of Agave mexicana. Again, Neubergt has shown
that a number of substances, under the action of leht and in the presence of
an optical sensitiser, form this aldehyde; indeed it is probable that there are
in the plant many substances which under suitable conditions can give rise to
formaldehyde or to hydrogen peroxide.
Summary.
1. The photo-chemical development of formaldehyde, which has been
observed to occur in films of chlorophyll extract in contact with air containing
carbon dioxide and water vapour, is due solely to the decomposition of the
films under the action of the oxygen of the air. No formaldehyde is produced
when such films are illuminated in a moist atmosphere of nitrogen or of
carbon dioxide. There is thus at present no evidence for the photo-synthesis
of the aldehyde from carbon dioxide by chlorophyll outside the plant.
2. The above oxidation is accompanied by the bleaching of the films, and
appears to be effected by the action of hydrogen peroxide, in the formation of
which carbon dioxide can have no share, since there is no decolorisation in
moist carbon dioxide free from oxygen.
3. The bleaching (oxidation) of films of carotin extract is also associated
with the production of formaldehyde.
Since the experiments are being continued along the lines indicated, a full
discussion of the results obtained is for the present deferred. In conclusion,
the author wishes to express his indebtedness to Prof. V. H. Blackman,
at whose suggestion this work was undertaken, for his very valuable help
throughout the course of the research.
* ‘Biochem. Zeitschr.,’ vol. 57, p. 95 (1913).
+ ‘Comptes Rendus,’ vol. 150, p. 529 (1910).
t ‘Biochem. Zeitschr., vol. 13, p. 305 (1908).
386
The Action of Light on Chlorophyll.
, By Haroip Wacker, F.R.S.
(Received February 6,—Read March 5, 1914.)
The chemical changes brought about by light in the green leaf leading to
the production of sugars and starch from carbon dioxide and water are still
far from being clearly understood.
To what extent the chlorophyll takes part in this process, whether it simply
performs the function of bringing the rays of light into contact with the
carbon dioxide and water in such a way as to enable them to effect a
synthesis of these two compounds, or whether the chlorophyll itself initiates
these changes by its own chemical decomposition, are problems still unsolved.
It is a well-known fact that solutions of chlorophyll in the presence of
oxygen become decolorised by light, and Pringsheim showed that the chloro-
phyll in a living leaf becomes rapidly blanched when submitted to the action
of an intense light focused through a lens.*
The earliest observations on the destructive effect of light on chlorophyll
appear to be those of Sir John Herschel, who in a series of papers+ published
more than 60 years ago described many interesting experiments on the action
of the rays of the solar spectrum on the vegetable colours expressed from the
petals and leaves of plants. From these experiments he concludes that (1)
the action of light destroys colour, either totally, or leaving a residual tint on
which it has no further or much slower action ; (2) the action of the spectrum
is confined, or nearly so, to the visible rays, as distinguished from the ultra-
violet and ultra-red rays, which are ineffective; and (3) the rays effective in
destroying a given tint are, in a great many cases, complementary to the tint
destroyed. He pointed out that the green colouring matter expressed from
leaves and spread on paper shows, as in the elder, a maximum of action, as
indicated by the destruction of colour, in the red rays, from which the action
falls off rapidly with a slight intermediate maximum in the region of the
yellow, then falls off again, and about the termination of the green again
increases, reaching another maximum in the blue violet, after which it falls
off again, gradually, and ceases to be traceable as the termination of the violet
is reached. He points out that “photographie pictures may be taken readily
on such papers, half an hour in good sun sufficing ; but the glairy nature of the
juices prevents their being evenly tinted, and spoils their beauty.” He did
* *Pringsheim’s Jahrb.,’ 1881 and 1882.
+ ‘Phil. Trans.,’ 1840, 1842 ; ‘ Phil. Mag.,’ 1843.
The Action of Inght on Chlorophyll. 387
not experiment with chlorophyll in a state of purity, owing to the nicety
required in its preparation.
There is evidence to show that, under the influence of light, the chlorophyll
in a living cell is constantly being destroyed, but that under normal conditions
the leaves remain green, the chlorophyll being reconstructed as fast as it 1s
destroyed. Thus when leaves are exposed toa stronger light than usual, they
become paler in colour, probably owing to the fact that under these conditions
the chlorophyll is destroyed at a more rapid rate than it is reconstructed.
This is frequently observed in the leaves of shade plants when exposed to
bright sunlight, and is also observed in Alge such as Spirogyra which
accumulate near the surface of water in the intense light of the sun during
the summer months.
Thus Ewart* states that “when green leaves are exposed to sunlight, the
decomposition of the chlorophyll goes on more rapidly than its production,
though the amount of chlorophyll decomposed is insufficient to cause a
change in the coloration visible to the eye.”
Stahl came to the conclusion} that the exposure of leaves to direct sunlight
for several hours gave no indication of the decomposition of chlorophyll.
Keeble showed, however, that leaves exposed to bright sunlight gave a
weaker solution of chlorophyll in alcohol than similar leaves kept in the
shade.{ Many other experiments support this view, notably those of Ewart§
conducted on plants both in temperate and in tropical regions. Lubimenko|
has also shown that the quantity of chlorophyll in a leaf varies with the
intensity of the light.
It is usually assumed that the decomposition of chlorophyll is bound up in
some way with the photo-synthesis of CO, and water, but as it is usually
considered to be more or less indirectly one of the results of photo-synthesis,
a sort of by-product as it were, very little attention has, so far as I know,
been paid to the products of its photo-decomposition.
Timiriazeff pointed out that chlorophyll is a true optical sensitiser in that
it absorbs radiant energy, and is at the same time an absorbent of one or more
of the products by which the bleaching is then brought about. The function
of chlorophyll is to decompose carbon dioxide; the chlorophyll absorbs the
* “Jinn. Journ. Bot.,’ vol. 31 (1895-97). ;
+ ‘Ann, du Jard. Bot. de Buitenzorg,’ vol. 11 (1893).
{ ‘Ann. Bot.,’ vol. 9 (1895).
§ ‘Journ. Linn. Soc. Bot.,’ vol. 31; ‘Ann. Bot.,’ vol. 11 (1897) ; ‘Ann. Bot.,’ vol. 12
(1898) ; see also references in Pfeffer’s ‘ Physiology,’ Eng. Ed., vol. 1, p. 334.
|| ‘Ann. Sci. Nat. Bot.,’ 1908.
“| ‘Comptes Rendus,’ 1885, and ‘ Ann. Sci. Nat. Bot.,’ 1885 ; see also ‘ Roy. Soc. Proc.,’
1903.
388 Mr. H. Wager.
rays of greatest energy and transmits this energy to the molecules of carbon
dioxide.
Again,according tothe hypothesisof Usher and Priestley,* the photo-synthesis
of carbon dioxide and water is accompanied by the formation of hydrogen
peroxide, and it is this latter compound that brings about the bleaching of the:
chlorophyll. From what we know of photo-chemical activity in other organic
compounds, it would, however, not be unlikely that the rays of light absorbed
by the chlorophyll may bring about a chemical change in it which is itself
sufficient to initiate the series of chemical reactions resulting in the formation
of sugar and starch. Thus Hoppe-Seyler,t quoted by Loeb,i “ expressed the
idea that chlorophyll undergoes first a combination with H,CO3 which, under
the influence of light, falls apart in such a way as to yield chlorophyll (or the
catalyser contained therein), O2 and a third product, the latter being sugar or
a substance from which sugar may be formed.” ‘It is obvious,” says Loeb,.
“that Hoppe-Seyler’s idea represents that conception of the action of the
catalyser which is more and more supported by the facts.”
Hansen§ suggests that the chlorophyll is capable of forming an unstable
compound with carbon dioxide, and that it is then passed on to the plasma
of the chlorophyll grain to be converted into carbohydrate. Sir W.-N.
Hartley,|| in discussing this, says that it is, however, much more probable on
chemical grounds that the compound of chlorophyll with carbon dioxide is
entirely decomposed, first by the elimination of oxygen, and, secondly, by
the elimination of water, so that there are successively formed compounds.
of chlorophyll (1) with carbon dioxide ; (2) with formic aldehyde ; (3) with
glucose ; and, finally, starch, completely formed, is split off the molecule.
The Bleaching of Chlorophyll in Light.
Crude chlorophyll was obtained in the ordinary way by boiling leaves of
grass or other plants in water and then extracting with alcohol. Methylated
spirit may be used for this purpose, but it is more satisfactory to use
absolute alcohol. In order to obtain the chlorophyll in as pure a state as.
possible, the strong alcoholic solution was first filtered, then evaporated to:
dryness, and dissolved in petroleum ether. For many experiments ordinary
ether will serve, but for general use petroleum ether is to be preferred.
Paper tinged with chlorophyll, either in alcoholic solution or in petroleum
ether solution, was used, and also films of chlorophyll made by the evapora-
* * Roy. Soc. Proc.,’ vol. 77.
+ ‘Physiologische Chemie,’ p. 139, Theil I (1877).
t ‘Dynamics of Living Matter.’
§ ‘Bied. Centr.,’ 1888, see ‘Chem. Soc. Journ.,’ Abstracts, 1888.
|| ‘Chem. Soc. Journ.,’ 1891.
The Action of Inght on Chlorophyll. 389
tion of the chlorophyll solution on glass plates and in glass tubes and
flasks.
The bleaching of chlorophyll can be conveniently demonstrated by
exposing the half of a strip of paper tinged with chlorophyll to the light, the
other half being kept in the dark. In sunlight the bleaching takes place
very rapidly, but very slowly in diffused light.
The action of the different rays of the spectrum can be shown by exposing
a piece of paper tinged with chlorophyll or a glass plate covered with a
layer of chlorophyll to a sunlight spectrum, and it will be seen that the
bleaching takes place as described in Herschell’s experiments and more
recently by Reinke* and by Dangeardt in those parts of the spectrum where
the light is absorbed. A convenient method of showing the different effects
of the principal parts of the spectrum is to make use of filters through
which definite wave-lengths are transmitted. The Wratten and Wainwright
filters are suitable for this purpose, and the action of light is much more
rapid than with the pure spectrum. The disadvantage of filters is that the
different colours absorb varying proportions of the light which they are
supposed to transmit. Thus whilst a red filter may transmit 78 per cent. of
the light, a blue filter may transmit only 16 per cent. of it.
The tricolour set of filters supplied by Messrs. Wratten and Wainwright
divide the visible spectrum into three nearly equal parts—red, green, and
blue—with some slight overlapping, but as this green allows rather more of
the yellow and blue ends of the spectrum to pass than is desirable, it is
better to add to the green another one which limits its range. The different
parts of the spectrum transmitted through the three filters which I have used
are as follows :—
Red—Standard tricolour filter, \ 710-590.
Green—Standard tricolour plus green (two filters), about » 550-480.
Blue—Standard tricolour filter, \ 510-400.
The bleaching of chlorophyll takes place very rapidly through the red
filter, much more slowly through the green and blue filters. If, however,
the light is allowed to act for a longer time through the blue and green
filters, the bleaching then becomes as pronounced through the blue as
through the red filter. Thus in bright sunlight it takes approximately
8-10 times as long to bleach chlorophyll paper through the blue filter as
through the red. This seems to indicate that the different effects of the red
and blue ends of the spectrum are proportional to (1) the absorption of light,
* “Bot. Zeit.,’ 1885.
+t ‘Le Botaniste,’ 1912 and 1913.
390 Mr. H. Wager.
and (2) the energy coefficient of the different parts of the spectrum in which
the absorption bands appear. Kniep and Minder* have pointed out that the
effects produced in photo-synthesis are approximately proportional to the
relative energy absorbed.
The Photo-decomposition Products of Chlorophyll.
The following experiments show that in the decomposition of chloro-
phyll by light two substances are produced, one giving the reactions of an
aldehyde and the other an oxidising substance giving reactions with potassium
iodide, by which the iodine is set free :—
Experiment 1: A piece of paper, tinged with alcoholic solution of
chlorophyll, was arranged so that one-half of it was exposed to a good light,
the other being kept dark. The half exposed to the light became bleached,
and when placed in Schiff’s solution the exposed portion developed a
beautiful pink, the unexposed half remaining green with no pink coloration.
Experiment 2: If a piece of chlorophyll paper exposed to light as in
Experiment 1 is placed in a solution of potassium iodide, the half exposed
to the light becomes reddish-blue in colour, due to the liberation of the
iodine, which acts upon the starch contained in the paper. The reddish-blue
colour is probably due to the action of iodine upon starch in the presence of
an excess of potassium iodide, for when the paper is washed in water the
reddish-blue colour disappears and is replaced by the ordinary blue starch
coloration.
Similar reactions to those described in these two experiments were found
to take place when the paper itself was exposed to light without the chloro-
phyll, but the coloration was not so strong in either case.
Experiment 3: Two pieces of common note paper, similar to that used in
Experiments 1 and 2, were exposed to light in the same way. One was
placed in Schiff’s solution. The exposed half became distinctly pink. The
other was placed in potassium iodide and the exposed half became light
brown. This seemed to indicate that the coloration in both cases was due to
the paper and not to the chlorophyll.
Various kinds of paper were then experimented with, and it was found
that in all cases a reaction both with Schiff’s solution and with potassium
iodide occurred, but that in the case of good superfine note paper the
reactions were very slight. Accordingly, in all subsequent experiments with
chlorophyll-tinged paper, a superfine note paper was used.
Experiment 4: A strip of W. H. Smith and Son’s superfine cream laid
note paper was tinged with chlorophyll and exposed to light as in Experi-
) * ¢Zeit. Bot.,’ 1909.
The Action of Inght on Chlorophyll. 391
ments 1 and 2. This was then cut longitudinally into two, and the two
strips were then placed in Schiff’s solution and in potassium iodide solution
respectively. In both cases a strong reaction was obtained in those portions
exposed to light. On comparing the results with the same paper not tinged
with chlorophyll it was found that the reaction both in Schiff’s solution and
in potassium iodide solution was very strong with the chlorophyll-tinged
paper but slight and almost negligible with the plain paper. Prolonged
exposure of the plain paper to light gives a stronger reaction, but in no case
as strong as the chlorophyll-tinged paper.
It was important to determine whether the solution of chlorophyll itself
is able to give the reaction, apart from the paper. As alcohol gives a strong
reaction with Schiff’s solution it was necessary to dissolve the chlorophyll in
some other solvent. For this purpose petroleum ether is suitable, as it does
not give any reaction with Schiff’s solution or with potassium iodide solution
either in the dark or in the light.
Experiment 5: Four smali test-tubes were partly filled with a solution of
chlorophyll in petroleum ether and tightly corked. Two were exposed to the
light and two kept in the dark. When those exposed to light were con-
siderably decolorised, a small quantity of Schiff’s solution was added to one
and a small quantity of potassium iodide plus starch solution was added to
the other. These solutions did not mix with the petroleum ether, but on
shaking up the test-tubes the Schiff’s solution became bright pink, the
potassium iodide and starch solution became bluish-brown. The petroleum
ether solutions which had been kept in the dark were treated in the same
way with Schiff’s solution and potassium iodide starch solution respectively,
and in neither case was any reaction observed.
These experiments show clearly that the decomposition of chlorophyll is
accompanied by the formation of an aldehyde and of a substance capable of
oxidising the potassium iodide and setting free the iodine. It is extremely
interesting to find that the same reactions are obtained with some kinds of
paper when exposed to light. This is probably due to the decomposition of a
substance,in the paper the nature of which is unknown.
The same results are obtained when films of chlorophyll on glass are
exposed to the light, and it can be further shown that the oxidising substance
produced is a gas soluble in water.
Experiment 6: About 5c.c. of a strong petroleum ether solution is carefully
evaporated in a 50 c.c. flask so as to leave a thin film of chlorophyll on the
sides and bottom of the flask. The ether should be completely evaporated,
and a stream of air forced through the flask to remove all traces of the ether.
The neck of the flask should be surrounded with black paper. A few drops
392 Mr. H. Wager.
of distilled water are placed in the flask. A strip of potassium iodide paper
about two inches long is then attached to a cork, and the flask is corked up
so as to allow the strip of paper to hang down in the neck of the flask.
Another flask should be fitted up in precisely the same way but without
chlorophyll. Both flasks should now be exposed to the sunlight. The
bleaching of the chlorophyll takes place very rapidly. The strip of
potassium iodide starch paper becomes purplish blue in the chlorophyll
flask, showing that iodine has been liberated, but remains quite unchanged
in the control flask. The strip of potassium iodide starch paper is now
removed and a few more drops of distilled water are placed in the flask,
which is then corked and the contents well shaken up. The water in the
flask is then poured into two tubes. ‘To one of these a few drops of Schiff’s
solution is added and a pink coloration soon develops, showing the presence
of an aldehyde. To the second tube a few drops of a 10-per-cent. solution of
potassium iodide is added, and then on the addition of a freshly made starch
solution, a blue or reddish-blue coloration is obtained, indicating the presence
of an oxidising agent capable of setting free the iodine in the potassium
iodide.
If the bleaching has been continued long enough, the sides of the flask are
now covered with a thin white layer of a substance which should be well
washed to get rid of the remnants of the soluble aldehyde, and it will then
be found that this white substance is insoluble in either hot or cold water.
If, however, the bleaching is prolonged for a considerable time a much smaller
amount of the insoluble white substance remains.
If we expose the chlorophyll paper behind coloured filters, we find that
both the aldehyde reaction and the potassium iodide reaction are much
stronger in the red than in the blue and weakest in the green. If, however,
the exposure behind the green and blue filters is prolonged to about 8 or
10 times that of the red, the reaction in the blue becomes as strong as in
the red.
The reaction for aldehyde is therefore proportional to the bleaching effect,
and is approximately proportional therefore to the synthetic activity in the
different parts of the spectrum.
The longer the light is allowed to act, the more completely does the
chlorophyll become bleached, with a corresponding increase in the aldehyde
reaction. In the case of the potassium iodide reaction, however, the converse
is the case. When chlorophyll films, either on paper or on glass, are sub-
mitted to the prolonged action of light, the reaction with potassium iodide is
much weakened, and may be completely absent. The explanation of this is
probably that the oxidising substance is a volatile gaseous product, which
The Action of Inght on Chlorophyll. 393
tends to disappear as soon as it is formed, whilst the aldehyde is a solid which
remains in the paper or in the film left on the glass. But it is not impossible
that the oxidising substance may be of service in connection with the chemical
reactions that take place in the chlorophyll, and may become used up in
this way.
Instead of the extract of chlorophyll we may use dried leaves, or the
chlorophyll expressed from living leaves and spread upon paper. We may
also use layers of Luglena viridis, aloce and other green organisms spread over
the surface of paper. In all these cases we can get by appropriate treat-
ment, after exposure to light. both the aldehyde and potassium iodide
reactions.
We can also show that both these reactions take place actually inside a
leaf when the chlorophyll is sufficiently bleached. Thus if sunlight is
condensed by means of a lens upon a living Oxalis leaf which contains
abundance of starch, the chlorophyll in a small area of the leaf is bleached.
If the leaf is now treated with Schiff’s solution we get a strong aldehyde
reaction in the bleached part ; if treated with potassium iodide solution the
starch grains in and around the bleached area become coloured blue. The
last experiment is not an easy one to perform as it is very difficult to hit
just the right moment to stop the bleaching in order to get the potassium
iodide reaction.
Is Formaldehyde produced by the Photo-decomposition of Chlorophyll ?
The observations of Pollacci,* Usher and Priestley,t Harvey Gibson,t and
Schryver§ all show that formaldehyde is produced when chlorophyll is
exposed to sunlight in the presence of carbon dioxide but not in its absence,
or possibly in minute quantities only. It is therefore important to determine
whether the aldehyde produced in my experiments is composed of formaldehyde
or whether it contains formaldehyde. The test used by Harvey Gibson gives
a very pronounced reaction even when formaldehyde is present in quite small
quantities. I have obtained a reliable reaction with 1/1,000,000, and a very
pronounced reaction with 1/100,000. The test is carried out as follows :—
About 3 c.c. of pure concentrated sulphuric acid are placed at the bottom of a
small test-tube ; a few drops of a 5-per-cent. solution of gallic acid in absolute
alcohol are poured gently on to the surface of the sulphuric acid and the
liquid to be tested is then added; if formaldehyde is present, a beautiful
* ‘Inst. Bot. d. R. Univ. di Pavia,’ 1902 ; see ‘L’Année Biologique,’ 1903.
+ ‘Roy. Soc. Proc.,’ 1906.
{ ‘Ann. Bot.,’ 1907.
§ ‘Roy. Soc. Proc.,’ 1909.
394 Mr. H. Wager.
blue-green ring appears between the upper and lower liquids. Unfortunately
this test is not reliable, as dilute solutions of sugar and starch and various ©
other substances bring about the formation of a green or blue-green ring.
This may be due to the fact that the sulphuric acid decomposes such
substances as starch and sugar, and that a transitory product of this.
decomposition may be formaldehyde. Consequently, although the reaction
is extremely useful for purposes of preliminary test, it cannot be relied upon
to prove the presence of formaldehyde.
Rimini’s test, as modified by Schryver, is extremely sensitive to form-
aldehyde and will easily detect 1/1,000,000. Here, however, the presence
of various substances in the crude chlorophyll seems to interfere with the
reaction, as shown by Schryver, and I have not been able to satisfy myself
that the colour reaction given by this test with solutions of bleached chloro-
phyll is due to formaldehyde. Colour reactions are obtained which seem to
indicate that formaldehyde is present in films exposed to light both in the
presence and in the absence of carbon dioxide, but the reaction varies con-
siderably with certain limits. Sometimes a dirty orange colour is produced,
which is nothing like so distinct as the colour obtained with 1/1,000,000 of
formaldehyde, sometimes a deeper coloration which more nearly resembles.
the formaldehyde coloration, but is more of an orange red or deep orange
than the bright, clear red of the formaldehyde reaction. In any case, none of
my experiments shows more than a very small quantity of formaldehyde in this.
way, although the reaction given by Schiff’s test in all cases indicated a much
larger amount of aldehyde. For example, a solution of bleached chlorophyll
showed a reaction for aldehyde with Schiff's solution equal to more than
1/25,000, but on testing the same solution with Rimini’s test, the result
showed the presence of certainly not more than 1/1,000,000 of formaldehyde.
It appears to me from a large number of experiments that, although the
aldehyde in the bleached chlorophyll may contain a small quantity of
formaldehyde, the major part of it consists of some other aldehyde, the
nature of which I have not been able to determine.
The Oxidising Compound of Chlorophyll.
The gaseous oxidising substance formed on exposure of chlorophyll to light
is soluble in water. The experiments of Usher and Priestley suggested the
possibility that it might be hydrogen peroxide. A solution was prepared
by exposing a film of chlorophyll on water to the light, which gave a strong
reaction with potassium iodide and starch, the iodine being liberated at
once and colouring the starch blue. Various well-known tests for hydrogen
The Action of Light on Chlorophyll. 395
peroxide were then tried, but all gave a negative result. The following are
some of the results obtained :—
Dilute solution of chromic acid with sulphuric acid. This gives a
distinct blue coloration with 1/1,000,000 of hydrogen peroxide, but no
reaction with the chlorophyll solution.
One of the most delicate tests for hydrogen peroxide appears to be that
given by Roscoe and Schorlemmer in their text-book. When hydrogen
-peroxide is added to a solution of potassium iodide ,and ferrous sulphate,
iodine is set free. Other oxidising agents have the power of liberating
iodine from potassium iodide, but not in the presence of ferrous sulphate.
I have obtained a distinct reaction with 1/50,000 of hydrogen peroxide and
a reliable reaction with 1/1,000,000. In the presence of ferrous sulphate the
chlorophyll derivative gives no reaction, although the same solution gave a
strong reaction with potassium iodide and starch alone.
Leuchter’s test*: With this I obtained a very clear reaction with
1/500 hydrogen peroxide, but no reaction with 1/50,000. No reaction was
obtained with a bleached chlorophyll solution.
Titanium dioxide in concentrated sulphuric acid gives an orange-red colour
with 1/5000 of hydrogen peroxide, and a distinct yellow colour with 1/50,000 ;
no coloration was given with the chlorophyll solution.
Experiments were also tried with a solution containing ferric chloride and
potassium ferricyanide. This gives a precipitate of Prussian blue with
solutions of hydrogen peroxide; solutions of the chlorophyll derivative only
give a greenish yellow colour.
All these tests indicate, therefore, that the chlorophyll derivative is not
hydrogen peroxide.
The experiment was then tried of exposing a film of chlorophyll in the
dark to the action of a 20-per-cent. solution of hydrogen peroxide. If, as
Usher and Priestley state, the decolorisation is brought about by hydrogen
peroxide, we ought to get a very pronounced effect with so strong a solution.
The experiment, however, showed that even after ten days’ exposure to the
hydrogen peroxide, the chlorophyll was far from completely bleached, and
was still of a yellow or yellowish green colour. The experiment was tried
many times in different ways, but always with the same result. The
bleaching of chlorophyll in the light appears not to be due, therefore, to the
action of hydrogen peroxide, and the most probable explanation seems to be
that the light absorbed brings about a combination of the chlorophyll with
oxygen resulting in the formation of an organic peroxide.
It is interesting to note that other colouring matters react to light in the
* Chem, Zeit.,’ 1911, see ‘Chem. Soc. Journ.,’ Abstracts, 1911.
VOL, LXXXVIL—B, 2G
396 Mr. H. Wager.
same way with the formation of an oxidising substance capable of bringing
about the liberation of iodine from potassium iodide. Thus, if strips of
paper are soaked in solutions of the following dyes—methyl violet, methyl
green, eosin, fuchsin, and fluorescein, and are then exposed to light and
afterwards treated with a 10-per-cent. solution of potassium iodide, the
iodine is liberated and the starch contained in the paper is coloured blue or
reddish blue, a strong reaction being obtained in all cases. Cyanin, on the
other hand, although readily bleached by the light, does not give this
reaction.
Experiments made with narrow glass tubes lined with a thin layer of
methyl violet and eosin show, on exposure to light, that, during the process
of bleaching, oxygen is used up, but this is not the case with cyanin, which
becomes completely bleached without any appreciable rise of water in the
tube. In the case of methyl green and eosin, the absorption of oxygen
does not take place as rapidly and is not so pronounced as in the case of
chlorophyll.
The Photo-decomposition of Chlorophyll in a Brown Sea-weed—Laminaria.
In order to make experiments on the chlorophyll contained in the brown
sea-weeds, I collected pieces of fresh fronds of laminaria on the sea-shore and
brought them home wrapped in pieces of ordinary white paper. On removing
the paper, I found a blue coloration here and there where the paper had been
in close contact with the fronds. It was obviously the blue starch coloration
due to iodine. I at first thought that it might be due to chlorine,* possibly
contained in the paper, acting upon an iodine compound in the sea-weed and
causing the liberation ofiodine. A statement in Pfeffer’s ‘ Physiology,’ however,
led me to suspect that the coloration might be due to free iodine given off
by the laminaria itself. To test this, I took some fresh pieces of a frond otf
laminaria and placed them in a dilute starch solution free from chlorine. The
solution became coloured blue, showing quite clearly the presence of free iodine.
The colour disappeared again: in a very short time, much more rapidly, so it
appeared to me, than it would have done in a starch solution coloured by an
ordinary solution of iodine. I accordingly tried the experiment again, and
found on comparing it with a starch solution coloured with iodine to the
same depth of colour, that the laminaria solution lost its colour several hours
before the other. This indicated that the iodine was taken up again by
the laminaria from the starch solution, and it occurred to me that this might
be due to the slime which is secreted by the laminaria and which was found
* Chlorine is used in the bleaching of paper.
The Action of Inght on Chlorophyll. 397
in large quantities in the solution. I therefore placed equal quantities of a
light blue iodine-coloured starch solution in two test-tubes. To one of these
I added distilled water; to the other an equal quantity of the slimy liquid
obtained by soaking pieces of the frond of laminaria in water. The colour
disappeared at once, on shaking up, in the tube containing the slime, but not
in the tube to which distilled water only had been added. I then placed equal
quantities of a very dilute iodine solution (iodine in potassium iodide) in two
test-tubes. To one I added, as before, distilled water, to the other an equal
quantity of the slimy liquid from laminaria. These were then shaken and
left to stand for a short time. Equal quantities of a dilute starch solution
were then added to each, with the result that the blue coloration appeared
in the tube to which distilled water had been added, but no coloration at all
in the tube containing the slime.
These experiments show, therefore, that iodine is absorbed by the laminaria
slime, probably forming an additive compound with it, and it appeared
probable that a much more satisfactory iodine reaction would be obtained
with laminaria if the frond were first of all freed from slime by washing well
in water. I obtained the reaction very readily on a bright spring morning on
the sea-shore by placing pieces of the fresh frond free from slime in contact
with starch paper. The reaction is, however, very unequal; all parts of a
frond are capable of giving it, but not necessarily at the same time. The
most vigorous reaction was obtained in the growing region of the frond, the
swollen portion where the frond joins the stipe. The reaction appears to be
associated with those layers of cells which contain the chlorophyll and the
brown colouring matter. Sections of the stipe and of the thicker portions of
the frond, when placed in contact with starch paper, showed a blue layer all
round where the paper had been in contact with the peripheral chlorophyll-
containing cells, and sections from the thinner portions of the frond also
gave the same result.
Pieces of the frond kept in sea-water in the dark gave no reaction, or at
times a slight one; a strong reaction was obtained when the fronds had been
exposed to a good light. The presence of the slime, however, may prevent
the reaction. So long as any of the brown colouring matter is present, the
iodine reaction may be given, but it is entirely absent in those parts of the
frond which have lost the brown colouring matter and show a green colour.
Whether the iodine reaction is associated with the brown colouring
matter I cannot say, but if one half of a piece of frond is placed for a short
time in hot water to destroy the brown coloration, the iodine reaction is
obtained only with the brown portions of the frond.
It is probable, therefore, though not quite certain, that the action of light
398 Mr. H. Wager.
on the chlorophyll of laminaria brings about the production of an oxidising
substance capable of effecting the decomposition of iodine compounds which
may be contained in the chlorophyll cells of the plant, and that the iodine
thus set free may either escape or be re-absorbed by the slime which occurs
in such abundance in laminaria.
The Photo-decomposition of Chlorophyll takes place only im the Presence of
Oxygen.
Three test-tubes were taken and lined with a film of chlorophyll by the
careful evaporation of a petroleum ether solution of grass chlorophyll.
One was placed with its open end in a strong solution of potassium hydrate
and pyrogallol, to absorb the oxygen; the second was placed in a strong
solution of potassium hydrate to absorb the carbon dioxide, and the third
was placed in distilled water. All three were kept in the dark for
24 hours, and were then exposed to the light. The second and third
bleached very rapidly. The first, which contained no free oxygen, remained
unbleached even after an exposure of some months. At the time of
writing, it has been exposed for four months in a south window and is
still unbleached. The second and third gave strong reactions both for
aldehyde and for the oxidising agent. The experiment shows quite clearly
that the bleaching of chlorophyll is the result of oxidation brought about
under the influence of light.
If this is correct, it occurred to me that we ought to be able to show that
oxygen is absorbed in the process. To test this, I obtained four tubes
16 cm. long and 4 mm. in diameter; they were drawn out at one end
to a point, which was then broken off, so as to leave an opening less than
1 mm. in diameter. Three of these were lined with chlorophyll from a
petroleum ether solution. Care was taken to get rid of all traces of the
ether by forcing a stream of air through the tubes for some time. I found
a bicycle pump useful for the purpose. The fourth tube contained no
chlorophyll and was simply used asa control. The three chlorophyll tubes
and the empty tube (No. 4) were then placed with their open ends down-
wards in distilled water contained in two separate beakers. The water
was at a sufficient depth to allow of its entry into the tubes to a height
of 2cm. This was done to allow of the expansion of the air in the tubes
when they were exposed to the heat of the sun. The upper narrow ends
of the tubes were then sealed with the bunsen flame. They were all
placed in the dark for 12 hours. The next day they were all carefully
measured, and it was found that the water was at the same height in
each tube. One of the chlorophyll tubes and the control tube were then
The Action of Inght on Chlorophyll. 399
exposed to sunlight. They were kept under careful observation to see
that the expansion inside the tubes did not drive out any of the air.
The chlorophyll in the chlorophyll tube soon began to bleach, and the
level of the water began to rise and, in the course of a few hours, it
reached a height of rather more than 1/5th of the tube, showing that
part of the air, probably the oxygen, had been absorbed. The water in
the control tube did not rise. The tubes were allowed to remain in the
light until no further rise in the chlorophyll tube took place. At this
stage the chlorophyll was not completely bleached, but as on prolonged
exposure to sunlight no further bleaching took place, it seemed fair to
conclude that all the oxygen had been used up. The tubes were now
brought to the back of the room into diffused light and allowed to stand
for some hours. Careful measurement of the chlorophyll tube showed
that the level of water in the tube had risen to a height corresponding
exactly to the percentage of oxygen likely to be present in the air
enclosed in the tube when the experiment started. To test this, the control
tube was placed in a strong solution of potassium hydrate and pyro.
This gradually diffused into the water contained in the tube and then
gradually absorbed the oxygen in the tube. In the course of 24 hours,
the level of this solution had risen in the tube until it was stationary,
and this was found to be almost exactly the same height as the level of
the water in the chlorophyll tube. This showed pretty conclusively that
in the bleaching of the chlorophyll the whole of the oxygen of the air had
been used up. To show that no oxygen was left, however, the chlorophyll
tube was itself placed in the potassium hydrate pyro solution; the tube
was gently warmed until the water was nearly driven out, and then, on
cooling, the pyro solution entered the tube and rose to exactly the same
level as the level of the water previously contained in the tube, and
remained at that level, thus showing that no oxygen had been left in the
tube.
Experiments were then made with the other two tubes which had been
kept in the dark; one was placed in a solution of potassium hydrate and
pyro, the other was placed in the sunlight for some hours. On leaving
them to stand for some hours at the back of the room to equalise the
temperature, the level of the liquid in both was the same. Further
experiments conducted more carefully with due attention to the corrections
necessary for temperature and pressure showed quite conclusively that
oxygen is absorbed when chlorophyll is bleached in the light, and that if
sufficient chlorophyll is present, the whole of the oxygen in the air in
contact with it is used up. It is possible, in fact, to make use of
400 Mr. H. Wager.
- chlorophyll instead of pyrogallol and potassium hydrate in making
quantitative determinations of the amount of oxygen contained in the air.
The Presence of Carbon Dioxide is not Necessary for the Photo-decomposition
of Chlorophyll.
The changes described in the last section are brought about just as rapidly
and as completely in the absence of carbon dioxide as when it is present.
Thus, two flasks were prepared with films of chlorophyll from a petroleum
ether solution of grass chlorophyll, as nearly as possible similar to each other.
Into one 2 c.c. of distilled water were placed together with a short tube con-
taining a stick of potassium hydrate to absorb carbon dioxide, and then
tightly corked with a strip of potassium iodide starch paper in the neck of
the flask. Into the second flask was placed 2 c.c. of water containing carbon
dioxide from a sparklet apparatus, and this was then corked up, also with a
strip of potassium iodide starch paper. Both were then exposed to sunlight,
and it was found that the bleaching was equally rapid in both cases, that the
potassium iodide starch paper was discoloured in both to the same extent,
and that the aldehyde in both was similar in amount, so far as could be judged
by the depth of colour produced with Schiff’s solution. The experiment was
tried several times under varying conditions, but the result was always the
same, the presence or absence of carbon dioxide made no difference in the
effects produced by the light.
But although carbon dioxide is not necessary for the photo-decomposition
of chlorophyll, it is possible that, when present, it may be used up in some
way corresponding to the photo-synthesis in the living plant. Experiments
conducted with known quantities of carbon dioxide in contact with thin
chlorophyll films in long narrow tubes, as used in previous experiments,
showed, however, that, whether present in large or in small quantities, the
carbon dioxide is apparently not used up in the bleaching of chlorophyll
outside the plant. The bleaching takes place quite readily so long as oxygen
is present, but the subsequent tests showed no diminution in the amount of
carbon dioxide, so far as this could be measured with caustic potash.
The experiments are not conclusive, however. It is possible that a very
small amount of carbon dioxide, too small to be measured quantitatively by
the somewhat rough methods at my disposal, may be used up, but the fact
that carbon dioxide is certainly not necessary for the bleaching of chlorophyll
or the production of aldehyde, and that, so far as my experiments go,
no appreciable amount of carbon dioxide is used up even when present
in considerable quantities, would seem to indicate that, under the conditions
of my experiments, carbon dioxide is not used up by the chlorophyll when
The Action of Light on Chlorophyll. 401
bleached in the light. It is important, however, that further experiments
should be made in which the carbon dioxide determinations can be made
more accurately.
The Photo-decomposition of the Green and Yellow Pigments of Chiorophyll.
The green and yellow pigments were obtained by shaking up an alcoholic
solution of grass chlorophyll with benzene. The alcoholic solution of the
yellow pigment was then evaporated to dryness and extracted with
petroleum ether. The benzene solution of the green pigment was treated in
the same way. Thin films of these two colouring matters were then
exposed to light (@) in the absence and (0) in the presence of carbon dioxide.
In both cases the yellow pigment bleached rapidly, and gave a very strong
reaction both with Schiff’s solution and with potassium iodide. The green
pigment bleached much more slowly and did not give quite as strong
a reaction with either Schiff’s solution or potassium iodide. Similar results
were obtained with strips of paper tinged with the green and yellow
pigments respectively. Thus, paper tinged with yellow pigment from grass
chlorophyll gave, after 40 minutes’ exposure to diffuse sunlight in January,
a strong reaction both with Schiff’s solution and potassium iodide. The
green pigment under the same conditions gave no reaction. Paper tinged
with ordinary grass chlorophyll gave a slightly stronger reaction than the
yellow pigment. At the end of two hours the green pigment gave a very
slight reaction with Schiff’s solution, but a strong reaction with potassium
iodide, the yellow pigment a strong reaction in both cases.
The more rapid oxidation of the yellow pigment can also be seen by
lining narrow glass tubes (a) with the yellow and (b) with the green
pigment. These are then placed with their open ends downwards in water
and exposed to bright sunlight. The water rises very rapidly in the tube
with the yellow pigment, showing a rapid absorption of the oxygen, but
more slowly in the tube with the green pigment. In both cases, however,
the whole of the oxygen in the tube ultimately becomes used up, and the
water rises to the same level in each.
The tubes were 39'4 cm. long. After exposure to light the water rose
86cm. The height of the water in a control tube of the same length was
0°5 cm. Consequently on subtracting this both from 39-4 and 8:6, the ratio
81 to 389 gives 20°82 as the percentage of oxygen absorbed.
The Action of Oxidising Agents wpon Chlorophyll.
As the decomposition of chlorophyll by light appears to be an oxidation
process brought about by the oxygen of the air in the presence of light,
402 Mr. H. Wager.
it occurred to me that similar effects might be brought about in the dark by
the use of some of the ordinary agents such as hydrogen peroxide and
permanganate of potash. I accordingly placed chlorophyll films, obtained
by the evaporation of a petroleum ether solution of grass chlorophyll, in
contact with a very dilute solution (pink) of permanganate of potash. These
were allowed to act for six days and were then examined. In all cases the
chlorophyll films showed considerable bleaching, and on carefully washing
them with water to get rid of the oxidising agents and then bringing them
into contact with Schiff’s solution, a pronounced pink coloration was pro-
duced, showing the presence of an aldehyde. The aldehyde at first appeared
in the film, but the colour soon became dissolved in the Schiff’s solution,
leaving a thin white layer in the glass. The powerful oxidising solution
made by adding a few drops of sulphuric acid to a dilute solution of perman-
ganate of potash acts very rapidly in bringing about the oxidation of
chlorophyll and the production of an aldehyde. A film of chlorophyll placed
in contact with the solution began to bleach at once, and in half an hour gave
a very pronounced reaction with Schiff’s solution.
The following experiments were also tried: A film of grass chlorophyll
placed in the dark in contact with a 20-per-cent. solution of hydrogen
peroxide for 16 days and then washed in water gave a strong reaction with
Schiff’s solution. The pink colour was first of all developed in the film,
but soon became washed out in the solution, leaving a whitish layer on the
glass.
A film of the yellow colouring matter of chlorophyll was treated in the
same way, and gave a similar reaction with Schiff’s solution.
A film of the green colouring matter of chlorophyll, treated in the same
way, showed very slight decoloration or bleaching, and gave no reaction
with Schiff’s solution.
Similar results were obtained when strips of paper tinged with chlorophyll
were used.
The bleaching of chlorophyll in the presence of hydrogen peroxide takes
place much more rapidly in the light than in the dark. Thus, a film of
erass chlorophyll was completely bleached in 12 hours in the light, but a
similar film was hardly changed after ten days in hydrogen peroxide in the
dark. In bright sunlight, complete bleaching was effected in three hours.
The film which had been bleached in the light gave a very strong reaction
for aldehyde, and the oxidation of the film was so complete that only a
trace of white film was left on the glass after the aldehyde had been
dissolved out by the Schiff’s solution.
The dilute sulphuric acid solution of permanganate of potash is a much
The Action of Inght on Chlorophyll. 403
more powerful oxidising agent than hydrogen peroxide in its action upon
chlorophyll. In the dark a very pronounced bleaching is obtained in half an
hour, and nearly complete decolorisation is effected in about two hours, with a
correspondingly strong reaction for aldehyde. In the light the bleaching
takes place slightly more rapidly than in the dark. :
The yellow colouring matter of chlorophyll bleaches very rapidly, the
green colouring matter very slowly in permanganate of potash and sulphuric
acid. Two films of equal size were prepared in two test-tubes, (1) of the
yellow colouring matter, and (2) of the green colouring matter of grass
chlorophyll, and equal quantities of the permanganate solution were poured
into each with the following results :—
(1) Yellow colouring matter: In 50 seconds the permanganate solution
was nearly colourless. At the end of two minutes it was poured off; the
film was quite bleached and gave a strong reaction for aldehyde with
Schiffs solution. The experiment was repeated with the yellow pigment
from leaves of Chrysonthemum parthenium (Keverfew) with a similar result.
(2) Green colouring matter: At the end of one hour the permanganate
solution was not quite colourless; the film still showed a yellow-green
coloration, but was more strongly bleached in the thinner parts. In order
to ensure more complete bleaching, fresh quantities of permanganate
solution were added from time to time, but even at the end of 12 hours the
bleaching was not complete. On testing with Schiff’s solution, the film
gave, however, a strong reaction for aldehyde.
None of the films bleached by oxidising agents, either in the dark or in
the light, gave a reaction with potassium iodide.
Experiments with strips of paper tinged with (1) grass chlorophyll, (2)
the yellow pigment, and (3) the green pigment from grass chlorophyll, gave
similar results on treatment with the permanganate solution. (1) and (2)
began to bleach at once, and at the end of half an hour gaye a strong
reaction for aldehyde; (5) showed a slight reaction only at the end of two
hours.
From these experiments we may draw the extremely interesting
conclusions that, so far as the production of an aldehyde is concerned, the
oxidation of chlorophyll in the dark by means of solutions of hydrogen
peroxide and permanganate of potash brings about a similar change to that
which is effected when chlorophyll is acted upon by light in the presence of
oxygen.
We have seen that the yellow colouring matter obtained both from grass
chlorophyll and from the chlorophyll extracted from the leaves of Chrysan-
themum parthenium bleaches very readily in the light, and also in
VOL. LXXXVII.—B. 2H
AOA Mr. H. Wager.
oxidising agents. This led me to suspect that the yellow colouring matter
extracted from etiolated leaves—leaves in which only a yellow colouring
matter had developed—would give the same results. The yellow-orange
colouring matter extracted from etiolated rhubarb leaves was found,
however, to bleach more slowly, both in light and in oxidising reagents,
than ordinary chlorophyll obtaimed from grass, and very much more slowly
than the yellow pigment from grass chlorophyll. Whether this has
anything to do with the lack of photo-synthetic activity which Miss Irving*
has found in chlorophyll not completely developed I cannot say, but,
considered in the light of Miss Irving’s observations, that the photo-synthetic
activity of chlorophyll does not reach its full strength until the chlorophyll
has been fully formed, the retardation of the photo-oxidation of the etiolin is
of considerable interest.
It is, of course, possible that the yellow colouring matters from other
plants may be found to behave differently in this respect, and too much
stress must not be laid, therefore, upon the experiments just described. It
is proposed to continue these observations.
The Action of Reducing Agents upon Oxidised Chlorophyll.
We have seen that the photo-oxidation of chlorophyll results in the pro-
duction of an oxidising substance and of an aldehyde. Both are therefore
oxidation products, and it was of some interest to ascertain the action of
reducing agents upon them.
Three strips of paper coloured green by grass chlorophyll in a petroleum
ether solution were exposed to the light until visibly bleached: 1 and 2
were then placed in a strong solution of phenylhydrazine, 5 was cut in two
and one portion was treated with Schiff’s solution, the other with a 10-per-
cent. solution of potassium iodide. Both gave a strong reaction. Aiter being
kept in the phenylhydrazine solution for three hours, 1 was placed in Schiff’s
solution, 2 in potassium iodide solution, and in neither case was any reaction
obtained. :
Similar results were obtained with stannous chleride, and with a pyro-soda
photographic developer.
Chlorophyll paper oxidised in the dark by the permanganate of potash
solution, then treated for three hours with phenylhydrazine hydrochloride also
gave no reaction either with Schiff’s solution or potassium iodide.
The reducing agents do not bring back the green colour to the oxidised film,
but the activity of both the products of chlorophyll photo-oxidation is
destroyed. - ;
* ‘Ann. Bot., 1910.
The Action of Inght on Chlorophyll. 405
Conclusion.
The experiments outlined in this paper indicate, so far as experiments con-
ducted on dead chlorophyll extracts can be taken as an indication of what
goes on in the living plant, that the bleaching of chlorophyll is not a result
of the activities set up by photo-synthesis, as suggested by Usher and Priestley,
but is the actual basis and starting point of the changes set up in the green
leaf under the influence of light. In other words, the aldehyde produced
under the conditions described in this paper is a product of the photo-
decomposition or photo-oxidation of chlorophyll and is not a result of the
direct photo-synthesis of carbon dioxide and water.
The aldehyde appears to be in fact purely a product of the photo-oxidation
of chlorophyll. This modifies our conception of the changes which may
possibly take place in the living plant. We know that carbon dioxide is
necessary for the production of sugar and starch in the living cell. But
if the sugar and starch are produced as the result of changes taking
place in an aldehyde, and if the aldehyde is a direct product of the decom-
position of chlorophyll, then we must conclude that the carbon dioxide before
it can be used is built up independently into the chlorophyll molecule, and
it is possible that the’ production of sugars and starch may be initiated by
photo-oxidation of the chlorophyll rather than by the direct photo-synthesis of
carbon dioxide and water.
Summary.
1. An account is given in this paper of some of the effects produced by
light upon chlorophyll. When chlorophyll is exposed to the light at least
two substances are formed, one of which is an aldehyde or mixture of alde-
hydes and the other an active chemical agent, capable of bringing about the
liberation of iodine from potassium iodide.
2. These products of decomposition can be very easily demonstrated by
means of strips of paper tinged with chlorophyll. When bleached in the light
and placed in Schiffs solution, a deep pink colour is developed showing the
presence of an aldehyde; but if placed in a 10-per-cent. solution of potassium
iodide, a reddish blue coloration, which becomes blue on washing in water, is
developed, showing the presence of an oxidising agent. The same products
are obtained when films of chlorophyll on glass are bleached in the light.
3. The bleaching of chlorophyll is less at the blue end of the spectrum than
at the red end, with a corresponding variation both in the aldehyde and
potassium iodide reactions. But if the exposure to the blue light is prolonged
to about eight or ten times that of the red light, the reactions are just as
pronounced. The bleaching and the corresponding products of decomposition
406 Mr. H. Wager.
are probably therefore proportional to the photo-synthetic activity of the
chlorophyll in the different parts of the spectrum.
4. The presence of formaldehyde is not very clearly indicated in my
experiments. Rimini’s test, as modified by Schryver, gives indications of a
trace of formaldehyde when chlorophyll is exposed to light both in the presence
and in the absence of carbon dioxide, but I do not consider the results reliable,
and in any case the reaction given is nothing like so strong as is indicated by
Schiff's solution. The test used by Harvey Gibson is also very sensitive to
formaldehyde, but is unreliable as it gives a pronounced reaction with
solutions of sugar and starch and other substances. All that can be said at
present is that in the photo-decomposition of chlorophyll a considerable
quantity of aldehyde is formed, with possibly a small amount of formaldehyde.
5. The oxidising substance appears not to be hydrogen peroxide, but it
may be an organic peroxide derivative of the chlorophyll.
6. The bleaching of chlorophyll in setw in dead green leaves, alge, and
other chlorophyll-containing organisms, gives the same products as the
chlorophyll extracts outside the plant.
7. Ifa fresh green leaf of Oxalis acetosella is exposed to an intense light
concentrated upon it by a lens, as in Pringsheim’s experiments, the bleached
chlorophyll gives an aldehyde reaction when placed in Schiff’s solution. If
the leaf contains abundance of starch, it may, after the action of the
intense sunlight, be placed in a solution of potassium iodide, when the
oxidising agent set free from the chlorophyll will liberate the iodine, and
the starch grains will be coloured blue. The experiment is not an easy one
to perform, as it is so very difficult to hit just the right moment to stop the
bleaching, in order to get the potassium iodide reaction. See also the
experiments on Laminaria.
8. The decomposition of chlorophyll with the production of aldehyde and
peroxide takes place just as readily in the absence of carbon dioxide as when
carbon dioxide is present. My experiments show that carbon dioxide is not
used up in the process even when present in considerable quantities. Carbon
dioxide is not necessary therefore to the production of the aldehyde.
9. The photo-decomposition of chlorophyll takes place only in the presence
of oxygen. Oxygen is used up in the process. If sufficient chlorophyll is
present, all the oxygen in the air in contact with the chlorophyll is absorbed.
Chlorophyll may be used instead of caustic potash and pyrogallol in the
analysis of air.
10. Chlorophyll is slowly oxidised in the dark by a solution of ip inode
peroxide. In the light the action is more rapid, but not more so than when
light acts on chlorophyll in the presence of oxygen. A rapid oxidation of
The Action of Inght on Chlorophyll. 407
the chlorophyll takes place in the dark in the presence of a dilute solution of
permanganate of potash to which a few drops of sulphuric acid have been
added. In both cases an aldehyde is produced which can be made evident
by means of Schiff’s solution.
11. Ifa strip of potassium iodide starch paper is exposed to light under
coloured filters the paper turns reddish blue under the blue filter, showing
the liberation of iodine, but not under the red filter. If, however, the
iodised starch paper is first of all tinged with chlorophyll and then exposed
to light under the same filters, a strong reaction takes place under the red
filter.
A strip of bleached chlorophyll paper, placed in contact with a strip of
damp iodised starch paper in the dark, is also capable of effecting the
liberation of iodine, and the starch paper turns blue.
12. It is suggested in conclusion that the production of sugars and starch
in the green leaf may be initiated by the photo-oxidation of chlorophyll and
the subsequent polymerisation of the aldehyde thus formed, rather than by
the direct photo-synthesis of carbon dioxide and water.
Intermittent Vision.
By A. Mattock, F.R.S.
(Received November 11,—Read December 11, 1913.)
[This paper is published in ‘ Proceedings,’ Series A, vol. 89, No. 612.]
Studies in Brownian Movement. 1.—On the Brownian Movement
of the Spores of Bacteria.
By Joun H. Suaxsy, B.Sc., and E. Emrys-Roserts, M.D.
(Communicated by Principal E. H. Griffiths, F.R.S. Received November 19,
1913,—Read January 29, 1914.)
[This paper is published in ‘ Proceedings,’ Series A, vol. 89, No. 614.]
RL in
mea “ON
MAY 28 1914 |
\ OF ra :
\Gfice Librat
VOL. LXXXVII.—B.
408
The Controlling Influence of Carbon Dioxide in the Maturation,
Dormancy, and Germination of Seeds.—Part LI.
By Frankuin Kipp, B.A., Fellow of St. John’s College, Cambridge.
(Communicated by Dr. F. F. Blackman, F.R.S. Received January 10,—
Read March 5, 1914.)
Introduction.
The cause or causes conditioning arrested development in moist seeds and
the nature of the impetus which results in germination are still in most
respects obscure. The problem of the non-germination of maturing seeds
while still upon the parent plant and the large range of cases of delayed or
non-germination of shed seeds which to all appearances are in good condi-
tions for germination form the basis of this research.
It is to be emphasised that the problem of seed dormancy is not limited
to the case of the dry seed. The more important, but less obvious, condi-
tions of dormancy are those found in moist maturing seeds, and in cases of
delayed germination in the presence of sufficient conditions of moisture and
temperature. It is these which have the most interesting analogies in other
fields, and an analysis of which may be more fruitful from the point of view
of physiology in general.
It is useful at the outset to examine certain conclusions that are being
reached by workers who have set themselves to elucidate the processes of
similar phenomena in other departments of physiology. In certain aspects,
the latency of the unfertilised ovum offers an analogy with the latency of
moist seeds. In each case the latency is only ended by the onset of definite
causes ; in each case in the absence of these causes the period of latency is
sooner or later terminated by death; and in each case also the sequence of
changes that follow the onset of the stimulus is, in a broad sense, physio-
logically comparable. The interest of this analogy, moreover, is increased
by the prominence which has recently been given to a simple interpretation
of the nature of the fertilisation stimulus. Loeb(1) has attempted to
outline its essential features as follows. These appear to be, firstly, an
acceleration of oxidations which follows destruction by cytolytic agents of
a cortical layer in the egg which has hitherto prevented oxygen from
reaching the surface of the egg and from penetrating into the latter
sufficiently rapidly. Secondly, Loeb believes that an internal change takes
place which renders innocuous the toxic products of oxidation. He shows
that the unfertilised matured ege dies soon, and he attributes this to the
Influence of Carbon Dioxide in Maturation, etc., of Seeds. 409
toxic action of products of oxidation, as its life can be prolonged in the
absence of oxygen.
Again, it has been a feature of recent work under many aspects to
emphasise the action of the ordinary metabolic products of cell life in
producing deep functional changes, both normal and abnormal. The nature
of the action of these products is being studied in detail, and it has
become clear in certain cases that what appears to be an act of excitatory
stimulus producing a certain forward change is in reality the removal
of a depressant stimulus normally present which acts as an inhibitant.
Thus, for example, it has been recently shown* that the growth of the
mammary glands in a pregnant female is due to a product of foetal growth
which acts by overcoming the inhibitory action of a substance which is
normally present and prevents the development of these parts.
The case of antithrombin normally present in the blood in sufficient
quantities to inhibit the action of any thrombin ferment formed, and so
preventing any intervascular clotting, is well known. The study of
immunity affords a very large number of instances of antibodies whose
function is the inhibition of the harmful stimulation of poisons. Czapek (3)
in his work on the anti-ferment reaction in tropistic movements of plants
has added another interesting example in this line of discovery. He
demonstrates geotropic stimulation to be accompanied by an accumulation of
homogentisinic acid due to the action of an antiferment inhibiting its break-
down by oxydase normally present.
In this paper the indicated problem of the dormancy of moist seeds has
been attacked from the point of view that dormancy must be conditioned by
the absence of an essential stimulus or by the presence of an inhibitory
agent. The two-sided question therefore which is presented at the
outset is as follows: What is the nature of the positive stimulus to
germination or what is the nature of the inhibition which must be over-
come to initiate this process ?
Influence of Carbon Dioxide in Inhibiting the Germination of Moist Seeds.
(a) Carbon Diomde Inhibits the Germination of Sceds without Producing
Injury.—\t will be useful to begin with a brief examination of the group of
phenomena classed under the term “delayed germination.” In one class of
cases it is known that many seeds do not immediately germinate in nature
even when to all appearance placed in optimum germinating conditions. This
is true of a number of native species which remain in the ground during the
winter, although freely germinating in the following spring. In another
* By Prof. Starling and Miss E. Lane-Claypon (2).
bo
H
bo
410 Mr. F. Kidd. The Controlling Influence of
considerable class of cases the seeds appear to be capable of remaining
indefinitely in the ground without germinating, while preserving latent their
power of growth under certain conditions, the nature of which does not
appear to be clearly understood. We find the embryos of these latent seeds
to be apparently in good germinating conditions, that is, supplied with
sufficient water, in an atmosphere containing the normal percentage of oxygen,
and at a temperature sufficient for germination.
In a large number of cases of this phenomenon quoted by Nobbe and
Hanlein (6), sporadic germination over periods of months, and even years, is
a marked feature. In natural conditions Srassica nigra is an example of
these cases of delayed germination. In Sussex it is locally called Kelke, and
every farmer and labourer along the northern slope of the South Downs will
give examples from his experience of the seeds sprouting in newly ploughed
land after they have lain dormant for years, while the land has been under
pasture or hay.
In certain of these cases of delayed germination in germinating conditions,
non-germination has been shown by Ewart to be accompanied by a lack of
water in the embryo due to the impermeability of the testa to water. These
cases do not bear upon our problem. It is with the range of cases in which a.
full water supply is demonstrated that interest lies. So far as explanations.
based on experiment have hitherto been forthcoming for non-germination in
these seeds, they have been mainly directed to elucidate this somewhat
striking phenomenon from the point of view that the testa is shielding the
embryo from a sufficient supply of oxygen.
Crocker (5) has reached this conclusion from his work upon the upper seeds
of Xanthium burrs, which normally do not germinate till after they have lain
over one year in the soil. He found that while at a temperature of 19° C.
these seeds would not germinate—though containing a sufficient supply
of H,O and though lying in a normal atmosphere (7c. with a partial
pressure of oxygen equal to 150 mm.)—germination could nevertheless be
immediately induced by removal of the testas. Recently Shull (4), working
upon these same seeds, has given us the actual minimum values of oxygen
necessary for the germination of the naked embryos. At a temperature of
21° C. the minimum partial oxygen pressure required by them is not more
than 12 mm. If we are to adopt Crocker’s view, therefore, that the non-
germination of these seeds with the testa intact is due simply to the fact that
only a subminimal quantity of oxygen can reach the embryo, we shall have to-
say that the wet testa is able to reduce the pressure of oxygen in its passage
through it from 150 mm. pressure to less than 12 mm.
It is conceivable that in the maturation of the seed and in delayed
Carbon Dioxide in Maturation, ete., of Seeds. All
germination under ordinary and special circumstances we may be dealing not
with an insufficient oxygen stimulus but with an inhibitory cause or group of
causes. Such a condition might result in the case of the seed if the testa acts
in any way as limiting the aération of the embryo, for we might expect then
two results :—
(1) A reduction in the amount of oxygen reaching the embryo, and
(2) A relative rise in the actual CO2 pressure in the embryo tissues.
The crucial question first arises, therefore, as to the actual effect of increased
pressures of CO: in the tissues of the embryo. The experiments which follow
have been immediately directed to ascertain in the first place the actual effect
of increased pressures of CO», upon the germination of quickly germinating
seeds. |
Technique of Experiments made to Ascertain the Effect of Increased Partial
Pressures of COz on Germination.—In setting these experiments a known
quantity of pure silica sand was first introduced into large flasks and
saturated with water. This was done by adding an excess of water and then:
drawing it off by tipping the flasks. If this was carefully done the sand
was left saturated with water in a layer adhering to the bottom of the
flasks. The seeds were carefully dropped on to this surface by means of a
glass tube, and, where necessary, as in the case of larger seeds, a further
measured quantity of H,O0 was added. The flasks were then stoppered with
new rubber corks fitted with one glass tube closed by means of pressure
tubing and a pinchcock. Gases in any proportion desired were now quickly
introduced by first withdrawing a quantity of air by an air pump, the
amount being read by a pressure gauge. Where small quantities of CO,
were desired up to 6 per cent. of an atmosphere, the operation was performed
by means of a specially made apparatus on the model of Hempel’s gas
burette, using mercury. By means of this apparatus very accurately
measured amounts of air can be withdrawn and equal lots of CO, introduced.
For higher percentages of CO, the air pump was employed. The artificial
atmospheres were for the most part checked by analysis after setting. The
carbon dioxide, oxygen, and nitrogen employed were in all cases from
cylinders as supplied by the Carbonic Acid Company and British Oxygen
Company.
412 Mr. F. Kidd. The Controlling Influence of
Table I—The Effect of Increased Partial Pressures of CO». on Barley
(Hordeum vulgare) in Retarding and Inhibiting Germination, and the
Resumption of Normal Activity on Removal of these Increased Partial
Pressures.
|
Germinations. | |
Actual percentage In presence of raised In air after removal of raised Final total
of CO, in air as set.| CO. pressures. After CO, pressures. After Der naee
(By analysis.) | of germina-
rs tion.
42 hrs. | 70 hrs.| 118 hrs. | 20 hrs. 44 hrs.| 70 hrs. Final
| | | | 12 days.
| | | |
0 7 9 10 | 100
(air with KOH)
so i £ 9 90
12°0 9 9 9 90
17°3 5 8 8 80
eae Z 6 Bi yaliyBicils Seneds ae 9 90
29-5 3 3 5 8 9 9 9 90
BOO- 1 Bll elo aio 10 100
Sie Ben 8 8 80
43°5 | Priel 1 : 3 e
96-0 | x
|
Temperature, 20° C. thermostat. 10 seeds in each experiment.
Table IIl.—The Effect of Increased Partial Pressures of CO» on Peas (Pisum
sativum) in Retarding and Inhibiting Germinations and the Resumption
of Normal Activity on the Removal of these Partial Pressures.
| Germinations.
Approximate In presence of raised In air after removal of Final total
| percentage of pressures of COs. raised pressures of CO. | germinations out
CO, in air as set. After After of five seeds in
(By analysis.) each case.
di 68 96 Tth | 8th | 9th | 11th | 20th
hrs. | hrs. | hrs. | day. | day. | day. | day. | day.
0 (air) 1 1 5 5 | 5
6 0) 2 3 4 A
12 1 3 ae is 5
18 1 5 | 5 5
24 10) a ary es) 5
30 3 4 | 4 5 5 5 5 5
50 3 4 5 5 5
70 2 4 4 4 4
100 0 1 2 3 3
Temperature, 20° C. thermostat. Five seeds in each case.
Carbon Dioxide in Maturation, etc., of Seeds. 413
Table II1I.—The Effect of Increased Partial Pressures of CO2 on Bean
(Vicia faba), Cabbage (Brassica oleracea), and Onion (Alliwm cepa) Seeds
in Inhibiting Germination, and the Resumption of Normal Activity on
the Removal of these Increased Partial Pressures.
Time during Resulli Final percentages
P . which seeds Diao ren subsequently in
ercentage of Sele : germination in ne
Species of | OO, in air in remain in artifi- artificial atmo- normal air.
ie oe 1 atmosphere at |
seed used. | which seeds were | “1% 20MOSphi sphere containing | =e
| set to germinate containing raised raised percentage |
8 “| percentage of ae Ou Of ger- | Of good
COs. 2 minations.| plants.
| days |
Cabbage 25 | lu | All inhibited 72 72
(50 seeds) 38 10 | * | 88 88
44 10 | Pe 76 76
to) 0) | Normal germina- | 84, 84
(air with KOH) |. | tion at once |
|
Beans 45 8 All inhibited 95 85
(30 seeds) 53 8 68 75 55
89 8 a | 85 50
0 0 Normal germina- 95 85
(air with KOH) | tion at once
Onion 23 11 28 per cent. ger- 44 44,
| (50 seeds) minated
| 30 11 All inhibited 50 50
68-7 11 Pe 46 46
1 (0) (0) Normal germina- 60 60
| | (air with KOH) | tion at once
Average temperature, 17 5° C.
(b) The Peculiar Case of White Mustard (Brassica alba).—Brassica alba
was peculiar among the seeds experimented on, in that inhibition was
continued indefinitely after the removal of the seeds from increased partial
pressures of CO, to normal air, and was then only terminated by the treat-
ments described in Tables IV and V.
White mustard seeds that have been inhibited by the action of CO2 while
germinating will lie indefinitely in germinating conditions without sprouting
or with sporadic sprouting over long intervals. They have all the appear-
ance of continued vitality, and they do not become attacked by moulds. The
part played by the testa in securing the continuance of the inhibitory effect
of carbon dioxide after the removal of the inhibitory agent is of great interest.
In the following table it will be seen that dormancy produced by CO,
was continued for two to three months after removal of the seeds to air,
suitable conditions for germination being maintained throughout. Finally
the seeds returned to normal activity and germinated 100 per cent. in every
case following the treatments described.
The Controlling Influence of
Mr. F. Kidd.
414
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416 Mr. F. Kidd. The Controlling Influence of
From the above tables of results it is clear that the case of Brassica alba
seeds is peculiar in that the inhibition extends after the removal of the
inhibiting CO. pressures from the atmospheres over the seeds. This after-
inhibition may extend for months accompanied by a sporadic germination.
The presence and condition of the testa seem to be the controlling factors in
this after-inhibition. Germination is at once induced by the removal of the
testa or usually by the complete drying of the seed.
(c) Delayed Germination in Nature-—F¥rom these experiments it is clear
that a condition is produced in the seeds of white mustard after treatment
with increased partial pressures of CO: which very closely parallels that of
seeds showing delayed germination in natural conditions. This fact is
brought out more clearly when the results obtained in the laboratory
with Brassica alba seeds are compared with the results recorded by Nobbe
and Hanlein (6) as occurring in nature. These authors give a large number ’
of cases in which they observed indefinitely delayed germination in natural
conditions accompanied by the sporadic sprouting of some of the seeds
extending over long periods. A few examples may be given.
Table VI.—Extract from Nobbe and Hanlein’s Tables of Seeds showing
Delayed Germination in Natural Conditions.
| Germinations after days.
} Number and name
of seeds. | | | |
5 ¢ | SB jie | ies 301 | 519 | 874 | Finally.
| | | |
_ 3 = =a
Capsella bursa-pastoris,| 3 6 _ _ TORS Ss 34°. | 58 aes
400 seeds | |
Thlaspi arvense ............ = | => | i Sao eee ei eo) 15 a7 7 gee |
| | |
A similar result was obtained by these authors with a large number of
species. The following may be mentioned :—Chelidoniwm majus, Myosurus
minimus, Plantago media, Potentilla argentea, Veronica beccabunga, Chenopodium
album, Campanula rotundifolia, Campanula persicifolia.
The similarity shown in the results obtained with the seeds of Brassica
alba inhibited under the influence of COs in artificial conditions to those
demonstrated by Nobbe and Hanlein (6) as occurring in natural conditions
is thus very marked.
Again, Crocker (5), working on a special case of delayed germination in the
upper seed of the burr of Xanthium, which, in contradistinction from the
lower seed of the burr, does not germinate in the first year after ripening but
in the second, found that by removing the testa he could induce immediate
Carbon Dioxide in Maturation, etc., of Seeds. A17
germination at any time after ripening. The case of the inhibited seeds of
Brassica alba offers an exact parallel to this case also.
(d) Experiments Reproducing in Nature, with CO. Naturally Produced, the
Results obtained in the Laboratory with Brassica alba Seeds—In drawing the
foregoing parallels, a reflection which is suggested is that the inhibition of
the Brassica alba seeds has been produced in the laboratory under conditions
remote from those found in nature.
The following series of experiments were therefore directed to ascertain
whether this objection is valid. The outcome of these experiments, it will
be seen, is to indicate that the results of inhibition under the influence of
CO, obtained in the laboratory with Brassica alba can be readily reproduced
in the soil in conditions such as may occur widely in nature. The method of
procedure was as follows:—Pits of various depths were dug in a garden soil
consisting of sandy loam with very few stones. Short, fresh-cut grass was
spread at the bottom in some cases. In others, green garden rubbish took
the place of grass. The earth was then returned to the pits, and seeds,
enclosed in small cotton-net bags, were inserted in it at various depths. The
CO: content of the atmosphere of this soil at various depths was taken
constantly during the experiments.
The following was a typical experiment:—On August 16, 1912, a pit
18 inches deep and 2 feet square was dug, a layer of packed green grass
about 3 inches deep inserted, and the pit then filled up by the return of
the earth removed. Seven days later, on August 23, three lots of 25 seeds
each were buried at depths of 3,6, and 9 inches in the earth in this pit
over the grass. At the same time three control lots of seeds were placed
at corresponding depths in a control pit close by, out of which the earth had
been dug, and similarly returned seven days previously, but in which no grass
had been placed.
The following are examples of the percentages of CO» found in samples of
soil air taken during the experiment at depths of 6 and 12 inches in the pit
containing grass :—
Per cent. Per cent.
August 23 at depth of 6 inches 12-4 CO2; at depth of 12 inches 18°8 COy.
nn SAS) A u 16°5 COz; i i 20:0 COs.
In the earth in the control pit, without grass, the CO. content of the soil
continued steadily at about 1 per cent. at depth of 12 inches.
After seven days in the soil the seeds were removed and examined. None
of those over the pit containing decaying grass had germinated at depths
of 6 and 9 inches, while at a depth of 3 inches only 3 out of the 25 seeds
had sprouted. All the seeds of the control lots at each depth in the pit
‘
418 Mr. F. Kidd. The Controlling Influence of
without grass had germinated and sprouted vigorously. The results
obtained are shown in the photograph and in the following table.
Results obtained with Brassica alba seeds in pit over Grass and in Control Pit
without Grass after seven days.
Table VII.—Results obtained with Brassica alba Seeds, planted (1) in Soil
over Decaying Green Grass, and (2) in Ordinary Soil.
Germinations out of 25 seeds after 7 days.
Depths.
Over decaying grass. Control in ordinary soil.
|
inches.
3 3 just sprouted 25 well grown.
6 0 25 ze
9 0 | 25 3 |
Thus 72 out of the 75 Brassica alba seeds planted in soil over decaying
grass were inhibited in conditions which may be supposed to occur some-
times in the soil (¢g., in the ploughing in of green crops*). These seeds
* The case of heavily dunged land would also suggest itself. Boussingault and Lewy,
in a large series of analyses of soil air, found 10 per cent. of CO, in manured soil 10 days
Carbon Dioxide in Maturation,.etc., of Seeds. 419
germinated sporadically afterwards, but systematic observations were hot
made in this first series of experiments as regards the after-behaviour of
the inhibited seeds. A further series was, however, set in which the
subsequent behaviour was noted. In this it was found that the results
obtained with seeds inhibited in the soil closely conformed in all respects
to the results obtained with those inhibited in laboratory conditions. In
this experiment, which was conducted at a temperature of 5-7° C., and in
which the seeds were left in the ground for 16 days, the CO2 content in the
soil over the buried grass rose from 10 per cent. on the 3rd day to 22 per cent.
on the 16th. No germinations occurred with the Brassica alba seeds placed
in the soil over the pit in which grass had been placed. All the seeds
placed in the soil in the control pit without grass vigorously germinated
within 10 days. When the inhibited seeds were removed to normal con-
ditions of germination, 20 per cent. germinated sporadically within the first
10 days. The remainder were apparently living at the end of two months.
None had been attacked by moulds. At this stage the testa was removed
from a number of the seeds, with the result that germination was imme-
diately induced, as in the laboratory experiments recorded above.
It would appear, therefore, that it is possible to reproduce in natural
conditions, which may occur widely in the soil, the results obtained in the
laboratory with inhibited Brassica alba seeds.
(e) Action of the Testa. Bare Embryos inhibited by Carbon Dioxide.—It is
desirable now to return to the problem in its original form, in which it was
indicated that germination may be due (1) to the action of a definite stimulus
such as would be supplied by the access of oxygen under suitable conditions
of moisture and temperature ; or (2) to the removal of some inhibitory agent
which has so far restrained the seed from entering upon the cycle of changes
which begins with germination ; or (3) to an inter-relation of both these
causes.
In the experiments with carbon dioxide acting on the seed in germinating
conditions so far related, it will be seen that certain partial pressures of CO,
have the effect of retarding and inhibiting germination, the see@ being
capable of resuming growth without any apparent injury on the removal of
the depressant. In the cases dealt with we seem to have two classes of
results which must be separated. In the cases of all the seeds excepting
after treatment. It appears from these results that caution is necessary in placing seed
in the ground into which green crops have been ploughed or which has been recently
heavily manured. In some of the experiments with pits described above the partial
pressure of CO, in the soil atmosphere over buried grass was found to be as much as
8 per cent. seven months after the green grass had been buried.
420 Mr. F. Kidd. The Controlling Influence of
Brassica alba it seems clear that the CO, has acted directly upon the tissues
of the embryo. On the removal of the CO: the seeds readily germinate. In
the case of Brassica alba the action of the carbon dioxide may have been the
same, but on the removal of the CO2 from the atmosphere the seeds do not
germinate but continue dormant. A direct action of CO. on the testa,
rendering it less permeable to the passage of gases, is suggested. Such a
change in the testa produced by CO2 would have two consequences: (1) a
reduction in the amount of oxygen reaching the embryo, and (2) a relative
rise in the COz pressure in the embryo tissues. The possibility thus arises
that lack of oxygen produced by a change in the permeability of the testa
due to the action of CO, has been the cause of inhibition in all the experi-
ments described. |
The following experiment was therefore made with Brassica alba seeds
from which the testas had been removed.
Table VIII.—Experiment indicating that Increased Pressures of CO: can
act Directly in producing Inhibition on the Naked Embryo of Brassica
alba.
Time seeds lay | Numbers and Percentage
Percentage | without germinating | condition of seeds of seed Porconteeeieae
2 in presence of high | set in presence of germinating eee savas
in alr. partial pressures high partial on removal to cane ae deel y
of COp. pressures of CO,. air. e
days.
60 2 10 without testas 100 (0)
80 7 10 55 100 30
The above experiment appears to demonstrate that the inhibitory action of
increased partial pressures of CO: may be direct upon the naked embryo of
mustard seeds. The phenomenon of prolonged after-inhibition did not occur
in these cases in the absence of the testa. Further experiments were made
with peas and white mustard with similar results. A conclusion, therefore,
which @ppears to be justified is that, while the inhibiting effect produced on
the embryo is the result of the direct action of CO: thereon, in the case of
Brassica alba an accompanying change in the testa plays an important part
in sealing the seed under the influence of CO, in a special dormant phase of
life.
Summary.
Experiments were conducted showing that the germination of seeds is
retarded or inhibited by high partial pressures of CO2 in the atmosphere.
Carbon Dioxide in Maturation, etc., of Seeds. A421
This retardation or inhibition produced by CO, was shown to be unaccom-
panied by injury. The seeds used in these experiments fall into two classes.
In the first class the seeds germinated at once after removal from the
inhibitory CO, pressures (beans, cabbage, barley, peas, onions). In the second
class the inhibition continued indefinitely after the removal of the inhibitory
COz pressures, and was terminated only by complete drying and re-wetting,
or by the removal of the testa. In this class a lowering of the degree of
permeability of the testa to gases by the action of CO, is indicated, a change
which would have two results: (1) a reduction in the amount of oxygen
reaching the embryo, and (2) a relative rise in the actual CO: pressure in the
embryo tissues. The condition of prolonged inhibition after removal to air
produced in Brassica alba is strikingly suggestive of the condition of seeds
often met with in nature, the germination of which is delayed in spite of
suitable conditions of temperature and water. The results obtained in the
laboratory with Brassica alba seeds were reproduced in the soil in natural
conditions by COs: arising from decaying vegetable matter. The high CO2
content of the soil air in these experiments was found to continue for a
considerable period. Attention was called to the importance of these facts in
agriculture.
LITERATURE CITED.
1. Loeb, ‘The Mechanistic Conception of Life,’ 1912.
2. Starling and Lane-Claypon, “An Experimental Enquiry into the Factors which
determine the Growth and Activity of the Mammary Glands,” ‘ Roy. Soe. Proc.,’
B, vol. 77, pp. 505-522 (1906).
3. Czapek, “The Anti-ferment Reaction in Tropistic Movements of Plants,” ‘Annals of
Botany,’ vol. 19, pp. 75-98 (1905).
4. Shull, “Oxygen Minimum and Germination of Xanthium Seeds,” ‘ Bot. Gaz.,’ vol. 52,
pp. 455-477 (1911).
. Crocker, “Féle of Seed Coats in Delayed Germination,” ‘Bot. Gaz.,’ vol. 42,
pp. 265-291 (1906).
6. Nobbe and Hanlein, “ Ueber Resistenz von Samen gegen die dusseren Factoren: der
Keimung,” ‘ Landw. Versuchs-Stat.,’ vol. 20, pp. 63-96 (1877).
or
422
The Functional Correlation between the Ovaries, Uterus, and
Mammary Glands in the Rabbit, with Observations on the
(Estrous Cycle.
By J. Hammonp, M.A., and F. H. A. MARSHALL, Se.D.
(Communicated by Dr. F. G. Hopkins, F.R.S. Received January 22,—Read
March 5, 1914.)
[Piates 17 anv 18.]
Recent experimental work has resulted in proving that there is a definite
functional correlation between the growth of the corpora lutea in the
ovaries and the hypertrophy of the mammary glands (Ancel and Bouin and
O’Donoghue). In the present paper experiments are described showing that
this hypertrophy in rabbits that have never been pregnant may be so con-
siderable as to lead to the production of milk, the secretion of which may
be temporarily increased by the injection of pituitary extract. Further
experiments are recorded showing that the uterus is not a necessary factor
in the development of the mammary gland. The influence of experimentally
produced corpora lutea upon the uterus is also described.
The Influence of the Ovarres upon the Mammary Glands.
It is well known that the mammary glands in man begin to undergo
enlargement at the time of puberty in correlation with the imcrease in
ovarian activity. Apart from this pubertal growth which is more or less
permanent, there is known frequently to be a slight swelling of the glands
at each menstrual period. A similar process takes place in the sow and
probably in other mammals at the “ heat” periods (Marshall). In the virgin
rabbit we have noticed a growth of the mammary ducts in six cases prior
to ovulation, but the cell proliferation, though quite definite, did not extend
to the glandular tissue. Experiments were undertaken to determine if the
growth could be increased by injecting foetus extract, with a view to bringing
further evidence to bear upon the hypothesis, put forward by Starling and
Lane-Claypon, that the anabolic changes involved in mammary hypertrophy
are dependent upon a fcetal hormone. The results, however, were negative
in each ease.
The following are the details of this series of observations. The extract
was made by grinding the fresh foetuses with sand and extracting with
Ringer’s fluid. The extract was then boiled and filtered. In the first three
experiments described below 39 rabbit foetuses were employed :—
Correlation between Ovaries, etc., in Rabbit. 423
(1) Fetus extract was injected for 15 days into a virgin rabbit aged
5 months. The rabbit was then killed, when it was found that the ovaries
contained a few follicles apparently ripe or nearly ripe, and the uterus a
few glands. The mammary development was limited to ducts which were
about 1 cm. long (fig. 1).
(2) In another virgin rabbit of the same age and treated identically the
ovaries showed a degenerate follicle and a few follicles apparently ripe.
The uterine glands were slightly developed, and the ducts of the mammary
glands were fairly well developed, being about 14 cm. long.
(3) Another virgin rabbit of the same age and treated identically gave
similar results to the last (No. 2).
(4) A virgin rabbit, aged 5 months, was allowed to undergo a sterile
copulation with a buck from which a portion of each vas deferens had been
removed. It was killed 12 days after copulation. Contrary to expectation,
no corpora lutea were found in the ovaries, but there was one large follicle.
The uterus contained a few glands, and the ducts of the mamme were about
4 em. long.
(5) A virgin rabbit, 7 months old, was allowed to undergo a sterile coition
with a vasectomised buck. It was killed 12 days afterwards. As in the
last case (No. 4) no corpora lutea were found, but protruding follicles were
present. The uterus had a few glands. The ducts of the mammary glands
were well developed, being about 24 cm. long.
(6) Another virgin, 7 months old, was allowed to undergo a sterile coition.
It was killed 24 days later. There were no corpora lutea, but many pro-
truding follicles, and the mammary ducts were about 2 cm. long.
It is thus seen that prior to ovulation the mammary development was
limited to a slight cell proliferation in the ducts, and that the growth was
not augmented by the injection of boiled foetus extract. On the other hand,
after ovulation (at least in the rabbit) definite mammary hypertrophy sets
in, as will be described below.
Probably in the majority of mammals ovulation takes place spontaneously
during cestrus. This is the case in the mare, the cow, the sow, the sheep
(at least ordinarily), and the bitch. On the other hand, in the rabbit, the
cat, and the ferret, ovulation, as a general rule, only occurs as a result of a
stimulus set up by sexual intercourse. To which of these categories man
belongs is still an open question.
It is generally believed that whereas the corpus luteum verum (or corpus
Juteum of pregnancy) and the so-called corpus luteum spurium' (which is
developed when pregnancy does not follow ovulation) are identical by origin,
the structure formed after ovulation does not hypertrophy to the same
VOL. LXXXVII—B. 2K
424 Mr. Hammond and Dr. Marshall. Correlation between
extent as when pregnancy supervenes, but on the other hand undergoes
retrogression after a few days. Ancel and Bouin, however, assert that in
such animals as the rabbit the corpus luteum, when formed, undergoes the
same amount of hypertrophy irrespective of the occurrence of gestation,
and that since these animals do not normally ovulate excepting after coition
the presence of corpora lutea is nearly always associated with the pregnant
condition. Further, they put forward the view, for which a considerable
body of evidence has been adduced, that in such animals as the rabbit the
corpora lutea provide the exciting cause for the growth of the mammary
glands during the first part of pregnancy.
In order to test this hypothesis they carried out experiments in which
the Graafian follicles of rabbits were ruptured under such conditions that
pregnancy could not supervene. The method usually adopted was to ligature
the vasa deferentia of the male rabbits. This operation although inhibiting
pregnancy, since spermatozoa cannct be ejaculated, does not prevent the
occurrence of coition. Since coition without seminal ejaculation is generally
sufficient to induce ovulation in the doe, corpora lutea could be formed just
as though pregnancy had supervened. Ancel and Bouin found that the
growth of the corpora lutea produced in this way was accompanied by a
hypertrophy of the mammary glands which continued for about 15 days or
until the corpora lutea began to undergo retrogressive changes. It was
naturally concluded that the growth of the mammary glands was brought
about by the activity of the corpora lutea. The further development of the
mammary glands in pregnant rabbits is ascribed by Ancel and Bouin to the
activity of a different gland, which is described as lying between the stroma
and muscular layers of the uterus, and is designated the myometrial gland.
Frank and Unger have described a case of a virgin rabbit with corpora
lutea in the ovaries and a breast development such as is usually characteristic
of the end of the first third of pregnancy.
Furthermore, O’Donoghue has investigated the relation of artificially
produced corpora lutea to the mammary glands. He took female rabbits in
a condition of cestrus, and ruptured the Graafian follicles mechanically.
In many cases corpora lutea were formed, and when this happened their
presence was associated with a growth of the mammary glands. The amount
of growth in 14 or 15 days is stated to have been about equivalent to that
shown by the normal pregnant rabbit in 12 days. If, however, the artificial
rupture of the follicles was not followed by the formation of corpora lutea
the mammary glands did not show any hypertrophy. O’Donoghue, had
previously adduced evidence that the corpora lutea and mammary glands are
functionally correlated in Dasyurus.
Ovaries, Uterus, and Mammary Glands in Rabbit. A425
The following is an account of our experiments. In Experiments 7-17
the animals were all virgin prior to the occurrence of the recorded coition.
The uterine changes are described separately below in dealing with the
question as to the influence of the corpora lutea upon the uterus.
(7) A rabbit, 6 months old, was killed 3 days after a sterile copulation
with a buck from which portions of each vas deferens had been removed.
The ovaries contained corpora lutea. The ducts of the mammary glands
were well developed, and there were slight traces of alveolar formation.
(8) A rabbit, 7 months old, was killed 5 days after a sterile copulation.
There were corpora lutea of two ages present in the ovaries, and the mammary
glands were well developed with the alveoli containing a secretion that
appeared to be milk.
(9) A rabbit; 8 months old, in which the Fallopian tubes had been
ligatured, was killed 9 days after a sterile copulation. The ovaries contained
corpora lutea. The alveoli of the mammary glands were in process of
formation.
(10) A rabbit, 8 months old, in which the Fallopian tubes had been
ligatured, was killed 12 days after a sterile copulation. Sections through the
ovaries showed that ovulation must have occurred in this case some
considerable time (probably about 25 days) previously, since the corpora lutea
were old and degenerate, and not recognisable on the surface of the ovaries.
The mammary glands showed signs of involution, but milk was present in
both the large and the small ducts. Milk could be expressed from the nipples
before killing. j
(11) A virgin rabbit, aged 7 months, was found to have ovulated spon-
taneously, this being very unusual in rabbits as above mentioned.* The
ovaries contained corpora lutea, apparently about 14 days old. There were
numerous alveoli found in the mammary glands (fig. 2).
(12) A rabbit, aged 15 months, from which portions of the Fallopian tubes
had been removed, was killed 16 days after a sterile coition. The alveoli
of the mammary glands were well developed.
(13) A rabbit, aged 8 months, was killed 24 days after a sterile coition
with a vasectomised buck. Old corpora lutea were found in sections
through one of the ovaries. The alveoli of the mammary glands were well
developed and active, containing a granular milky secretion. Milk could be
squeezed from the nipples. The milk was examined microscopically and
* This rabbit was ina cage with another female. Doe rabbits in a state of cestrus
when kept together have been observed to “jump” one another after the manner of cows
when on heat, and it is possible that the stimulus set up in this way may be sufficient to
induce ovulation.
Kee
426 Mr. Hammond and Dr. Marshall. Correlation between
stained with Sudan II, when fat globules were seen. The fluid when collected
had the appearance of ordinary milk, and yielded a floceulent precipitate when
treated with dilute acetic acid.
(14) A rabbit, 10 months old, was injected every day with boiled foetus
extract together with boiled placenta extract from the 11th to the
24th days, after a sterile copulation, the Fallopian tubes of the rabbit having
been previously cut and portions removed. The rabbit was killed on the
27th day. Old corpora lutea were found in sections through the ovary. The
alveoli of the mammary glands showed signs of atrophy, but the ducts and
some of the alveoli contained milk. Before killing, a serous milky fluid was
expressed from the nipples. The milk was tested as before, and found to
contain some fat and albumen.
(15) A virgin rabbit, 11 months old, was rendered sterile by the Fallopian
tubes being severed. It was then injected with boiled extract of uterus from
the 11th to the 24th days, after a sterile coition. The rabbit copulated again on
the 29th day, and was immediately afterwards killed. Old corpora lutea were
found in the deeper parts of the ovary and several apparently ripe follicles on
the surface. The mammary glands contained milk. Previously milk had
been expressed from the nipples on the 19th, 21st, 27th, and 29th days after
the sterile coition. The milk of this rabbit, collected in a test-tube, had the
appearance of normal milk, and samples under the microscope were seen to
contain globules of fat.
(16) A rabbit was injected with boiled uterus extract from the 11th to
the 24th day after a sterile coition. Milk was expressed from the nipples
on the 19th, 21st, 27th, and 29th days. The milk was collected and examined
as in the previous case (No. 15). The rabbit copulated a second time with a
vasectomised buck on the 29th day; 30 days later the rabbit copulated
again and on the same day milk was expressed from the nipples; 28 days
later milk was again expressed from the nipples. Next day the rabbit
copulated again. Then on the same day 1 c.c. of pituitary extract was injected
and the animal killed. The mammary glands were well developed, but
showed signs of involution. They were full of milk. The ovaries contained
old corpora lutea and ripe follicles.
(17) A rabbit was injected daily with boiled placenta and fcetus extract
from the 11th to the 24th day after a sterile coition. Milk was expressed
from the nipples on the 19th, 21st, 27th, and 29th days, and was collected
and examined as in the preceding cases. On the 29th day (June 16)
the rabbit again underwent a sterile copulation; 28 days later (July 14)
although not pregnant, the rabbit plucked its fur from its breast and made a
nest as if preparing for parturition. On the same day milk was expressed
Ovaries, Uterus, and Mammary Glands in Rabbit. 427
from the nipples. Two days later (July 16) the animal again underwent a
sterile coition. On the 17th day afterwards an attempt was made to express
milk from the nipples but none could be obtained. On the 22nd day a little
serous fluid was obtained, and on the 28th day a considerable quantity of fluid.
The same day (August 13) the rabbit copulated a fourth time; 24 days later
(September 6) no fluid could be expressed from the nipples, but two days
later (September 8) a few drops of serous fluid were obtained. The rabbit
copulated again (September 24) and is still alive.
(18) A multiparous rabbit underwent a sterile copulation with a vasec-
tomised buck. On the 15th day after copulation no milk could be expressed
from the nipples. On the 20th day milk could be obtained in considerable
quantity. On the 22nd day the rabbit was again on heat and after undergoing
copulation was killed. The mammary glands were found to be full of milk.
Old corpora lutea and numerous degenerate follicles were found in sections
through the ovary. °
(19) This experiment was with a multiparous rabbit and the result was
similar to that of the preceding experiment, there being no milk on the 15th
day, but some milk on the 20th and 22nd days, on the latter of which the
rabbit copulated again with a vasectomised buck (August 15). Twenty-
two days later (September 6) a few drops of milk were obtained, and two
days later quite a lot of milk was drawn off. The rabbit is still alive.
(20) This experiment was upon a multiparous rabbit which was exceptional
in that no milk could be expressed from the nipples at any time during the
period between two successive copulations with a vasectomised buck. After
copulating a third time fluid could be expressed from the nipples on the 22nd
day and on the 24th day.
(21) A multiparous rabbit was killed 17 days after a sterile copulation with
a vasectomised buck. The mammary glands were found to be well developed
but they contained no milk.
It is thus seen that in pseudo-pregnant rabbits (that is, in rabbits in which
corpus luteum formation followed upon sterile coition) milk first made its
appearance about the 19th day after copulation. At about this period the
mammary hypertrophy appeared to have become complete and retrogressive
changes set in, anabolism giving place to katabolism, at any rate to a consider-
able extent. These changes took place in the absence of any observed activity
on the part of the myometrial gland, and it must be assumed that this gland
is not an essential factor in mammary development. Moreover the immediate
secretion of milk in considerable quantity followed by the characteristic
changes in the tissue of the mammary glands could be induced by the injection
of pituitary extract in the same kind of way as in normal lactation.
428 Mr. Hammond and Dr. Marshall. Correlation between
The interval between two cestrous periods (that is the interval occupied
either partly or wholly by pseudo-pregnancy) was from 22 to 30 days, the
period of gestation in the normal rabbit being 30 days.
Whether or not the corpus luteum plays any part in mammary growth-or
secretion in the latter stages of normal pregnancy isa point which has not been
determined.
In normal pregnancy the development of the glands is undoubtedly greater
than anything that occurs in pseudo-pregnancy, and it would seem probable
that some further factor is concerned in bringing about this growth. This
factor is possibly to be sought for either in the placenta, as suggested by
Basch, or in the myometrial gland, as supposed by Ancel and Bouin. Never-
theless, it is clear that the presence of corpora lutea alone, apart from the
existence of any subsidiary factor, suffices to stimulate gland growth to such
a degree of completion as to result in the secretion of milk.
As mentioned already, Ancel and Bouin distinguish between the corpora
lutea of pregnancy and the so-called “ periodic corpora lutea” which only occur
in animals that ovulate spontaneously. The artificially produced corpora lutea
in the rabbit are regarded as belonging to the former kind. Moreover in those
animals (like the rabbit) which only ovulate after coition the interstitial cells
are supposed to take the place of the periodic corpora lutea. It may be doubted
whether the distinction made between the two kinds of corpora lutea by Ancel
and Bouin should be insisted upon. In the first place the corpora lutea are all
formed in precisely the same way from the discharged follicles, while according
to Biedl the ovarian interstitial cells in rodents arise from connective tissue
which grows inwards so as to fill up the cavities of degenerate follicles. Such
cells are designated by Seitz “theca lutein cells” since they arise in the
theca interna of the follicles, and subsequently develop into cells resembling
those of corpora lutea. Miss Lane-Claypon, however, states that the ovarian
interstitial cells are derived, like the follicular epithelial cells, from the
germinal epithelium.*
Furthermore, from the account given by Hill and O’Donoghue it would
seem that the corpora lutea in Dasyurus always undergo the same degree of
development irrespectively of the occurrenve of pregnancy. They describe an
animal as being seen to clean out its pouch for the reception of young, although
it had not become pregnant, thus showing that in Dasywrus the cyclical changes
of the sexual organs, which are apparently consequent upon ovarian changes,
may even extend to the instincts associated with parturition and the nursing
of the young, although pregnancy had not taken place.
* T have noted the presence of interstitial cells in the ovary of the rabbit prior to
the maturation of any follicles.—J. H.
Ovaries, Uterus, and Mammary Glands in Rabbit. 429
A case of a rabbit which prepared a bed for a litter and secreted milk at
the termination of the pseudo-pregnant period has been recorded above.
Cases have also been reported by various observers of similar instincts in
bitches, which have been described as making preparations for parturition and
secreting milk nine weeks after coition although they had failed to become
pregnant. Thus Heape records instances of bitches which had been “lined”
but had “missed” having pups, yet had secreted milk at the time when they
were due to whelp, in sufficient quantity to admit of their rearing litters
belonging to other bitches. Cases have also been recorded by Noel Paton.
Moreover, several such eases of bitches which did not conceive but yet have
afterwards yielded milk have been recently reported to the authors.
It is suggested that in these animals the building up of the mammary
glands and the resulting secretion of milk may have taken place in response
to a stimulus arising in corpora lutea which developed after cestrus and
possibly persisted for an abnormal length of time. If this explanation
is correct it is clear that no essential distinction can be drawn between the
corpora lutea of pregnancy and the periodic corpora lutea in regard to their
functional relation to the mammary glands.
Our observations lend no support to the theories of Starling and Lane-
Claypon, Foa, Biedl and Koenigstein, who have supposed that the mammary
glands are built up under the influence of a hormone arising in the feetus,
neither are they confirmatory of the view put forward by Halban, who
regards the placenta as a factor in mammary growth. Our experimental
results are, at first sight, somewhat difficult to reconcile with the facts
observed by Ott and Scott, and Schafer and Mackenzie, who found that
corpus luteum extract (like that of pituitary) when injected into the
circulation has an immediate galactogogue action. It must be borne in
mind, however, that the sudden injection of considerable quantities of corpus
luteum extract into the circulation is not a process which occurs in nature,
and consequently we might expect its effect upon the mammary tissue to be
different from that of small quantities of the problematical hormone when
continuously secreted over a long period.
The Effect of Hysterectomy without Ovariotomy.
Experiments were also undertaken to ascertain whether or not the uterus
is an essential factor in mammary growth. As already mentioned, Ancel
and Bouin have expressed the opinion that in the later stages of pregnancy
the myometrial gland of the uterus is an exciting cause in mammary
development. It occurred to us that it was possible that the uterus might
also be an essential factor in bringing about mammary development in the
430 Mr. Hammond and Dr. Marshall. Correlation between
earlier stages of pregnancy, and that the corpora lutea might be unable to
exert their influence upon the mamme excepting through the mediation of
the uterus. The changes which the uterus undergoes (to be described below)
as a result of the formation of the corpora lutea lent a certain amount
of evidence in favour of this view. It had, however, been shown that the
removal of the uterus in young rabbits has no effect upon the subsequent
growth of the ovaries, for animals so operated upon after becoming mature
are capable of copulation, ovulation, and the formation of corpora lutea just
as though they had not undergone hysterectomy ; but the effects (if any) of
the removal upon breast development were not recorded (Carmichael and
Marshall).
The following is an account of our experiments :—
(22) The uterus was removed from a virgin rabbit when 10 months old.
Subsequently the animal copulated and was killed 25 days after copulation.
No remains were found of the uterus or Fallopian tubes, and one ovary was
missing, presumably having become absorbed as a result of vascular inter-
ference at the time of the hysterectomy operation. The other was normal
and contained nine corpora lutea. The alveoli of the mammary glands
showed signs of atrophy, but it was clear that they had undergone a con-
siderable growth previously. Both alveoli and ducts contained a secretion.
(23) The uterus was removed from a virgin rabbit when 3 months old.
After it had reached maturity it was allowed to copulate several times, and
killed 12 days after the last copulation. One ovary contained four corpora
lutea, the other having undergone atrophy. No remains of uterus or
Fallopian tubes could be found. The mammary glands showed a great
development of alveoli but no milk was present (fig. 3).
(24) The uterus was removed from a virgin rabbit when 3 months old.
After it had reached maturity it was allowed to copulate several times, and
was killed 9 days after the last copulation. The left ovary contained
several corpora lutea. Small pieces of the Fallopian tube were found
attached to it. The right ovary had undergone partial atrophy presumably
as a result of vascular interference, and there was a small piece of the right
Fallopian tube with a cyst. The mammary glands were well developed, the
alveoli being filled with a secretion,
(25) The uterus was removed from a virgin rabbit when 3 months old.
The rabbit subsequently copulated. A little serous fluid could be squeezed
from the nipples on the 22nd and 27th days after copulation. The rabbit
copulated again on the 28th day (August 14). A few drops of fluid were
expressed 25 days afterwards (September 8), when it copulated again and
was immediately killed. The mammary glands were well developed. The
Ovaries, Uterus, and Mammary Glands in Rabbit. 481
alveoli and ducts were full of a milky secretion. Both ovaries contained
corpora lutea. There were no remains of tubes or uterus.
(26) The uterus was removed from a virgin rabbit when 3 months old.
On the 27th day after copulation (which took place when maturity was
reached) fluid could be squeezed from the nipples. On the 28th day the
rabbit was killed, when it was found that the alveoli of the mammary glands
were well developed. The ovaries contained corpora lutea. There was
a small piece of one Fallopian tube left.
(27) The uterus was removed from a virgin rabbit when 3 months old.
It reached maturity, copulated, as in the preceding cases, and was killed
17 days later. No remains of uterus or tubes could be found. The ovaries
contained eight (three and five) corpora lutea. The mammary glands were
well developed, the ducts and alveoli being filled with a secretion.
_ These experiments show that mammary development occurring in rabbits
asa result of the formation of experimentally produced corpora lutea takes
place independently of any uterine influence. Thus the uterus is not
a factor in mammary growth any more than in ovarian growth. The
experiments show further that the presence of one ovary, with its contained
corpora lutea, is sufficient to bring about the mammary hypertrophy.
The Influence of the Corpora Lutea wpon the Uterus.
It has been concluded by Fraenkel and others that the corpus luteum is
an essential factor in the fixation of the fertilised ovum to the uterine wall
and in the nourishment of the embryo during the first stages of pregnancy.
This conclusion is based on the results of ovariotomy during early pregnancy
and on a large number of control experiments. Whether or not the
evidence is sufficient to justify the theory being stated in precisely this
form, it would seem clear that the development of the corpus luteum is
functionally connected-with the contemporaneous hypertrophy of the uterine
wall during the first stages of gestation, since the raised nutrition of the
uterus is dependent upon the presence of the corpus luteum (Marshall and
Jolly). Ancel and Bouin state that in the case of the rabbit the non-pregnant
uterus undergoes hypertrophic changes when corpora lutea are developed.
This has been called in question by Dubreuil and Regaud, but Niskoubina’s
observations are confirmatory of those of Ancel and Bouin.
The following is an account of our observations upon the changes under-
gone by the non-pregnant uterus after ovulation consequent upon sterile
coition (excepting in the case of Experiment 11 where the rabbit had
ovulated spontaneously). The condition of the ovaries and mammary glands
has been already described. The numbers of the experiments provide a
432 Mr. Hammond and Dr. Marshall. Correlation between
means of identifying the individual rabbits previously referred to. The
respective ages of the rabbits, which prior to the occurrence of the recorded
coition were all virgins, have also been given above.
' (7) In a rabbit killed 3 days after sterile coition the uterine glands
were just commencing to undergo active growth.
(8) In a rabbit of 5 days the glands were considerably developed and the
muscular walls had undergone some thickening.
(9) In a rabbit of 9 days the process had been carried further (fig. 5).
(10) Ina rabbit of 12 days the uterine glands were more numerous and
smaller than those of No. 9. They were also more closely packed, and the
uterus showed congestion. The muscular walls were very thick. It is to be
noted again that the ovaries contained very old corpora lutea (see above).
(11) This rabbit had ovulated and corpora lutea were present, apparently
about 14 days old. The uterus showed a great development of glands which
were elongated and formed a spongy-looking mass at the base of the folds.
The muscular coat was thickened.
(12) In a rabbit killed 16 days after sterile coition the uterine glands
were enlarged and spongy-looking. The capillaries in the stroma between
the glands were distended. The muscular layers were very thick.
(13) In a rabbit of 24 days the uterine glands were smaller than those of
No. 12, but still very active. The folds of the mucosa contained a large
amount of extravasated blood, showing that the congestion had resulted in
a breaking down of the blood-vessels. The muscular coat was moderately
thick (fig. 6).
The changes outlined above presented an essential similiarity to those
described by Hill and O'Donoghue for the pseudo-pregnant marsupial .cat.
There is a strikingly close likeness between the appearances which we
have just described (as shown in sections through the rabbit’s uterus
during the successive stages) and the figures published in Hill and
O’Donoghue’s paper on Dasyurus. In view of this great similarity there
can hardly be reason to doubt that the changes which take place in the
rabbit’s uterus after sterile coition are physiologically homologous with the
changes which occur in the uterus of Dasyurus during the period of pseudo-
pregnancy. As will be shown subsequently the recognition of this fact,
which has not hitherto been pointed out, materially affects the views
entertained by the above-mentioned authors regarding the nature of the
homology between the cestrous cycle of the marsupial and that of the
Eutherian mammal.
Lastly, the hypertrophic changes which take place in the uterus during
pseudo-pregnancy are clearly comparable to those which occur in true
Ovaries, Uterus, and Mammary Glands in Rabbit. 433
pregnancy in association with the development of the embryo, whose
presence necessitates the maintenance of a raised nutrition on the part of
the organ which protects it and through which it derives nourishment.
That the corpora lutea are a factor in preserving this raised nutrition
seems to have been established beyond question.
The Cstrous Cycle.
According to Heape a period of five or six months (7.e. spring and summer)
is the usual duration of the sexual season in the domesticated rabbit.
Heape says further: “ No doubt if they are kept warm, carefully fed, and
their breeding carefully regulated throughout the spring and summer, they
may exhibit oestrus also in winter, but it must be recollected that here
we are treating of cestrus independently of pregnancy, which is a very
different matter.”
Our experience has been different from that of Heape, for many of our
rabbits, kept in hutches in an outhouse and without any artificial heating,
have bred in the winter months, though not with the same frequency as
in spring and summer. The following is a record :—
Percentage
breeding.
Of 12 rabbits which copulated about Dec. 14, 5 had young ............... 41°7
9» 24 33 Mar. 22, 14 Pe PN tc Ries cae 58°3
aera 5 5 May 18, 17 Sem mstshs Renner scant 81-0
» 8 » 6 June 14, 8 Both Be. Sreneaenivan cepts 100 0
Half of these rabbits had been treated with Yohimbine, administered by
the mouth for several days before copulation, but the drug, although in
other cases it caused a pronounced congestion of the uterus, did not
increase the breeding powers or affect the fecundity, as compared with the
other rabbits which were kept as controls.
Prof. Punnett, who has kindly suppled us with further information con-
cerning the recurrence of cestrus in rabbits, finds that when kept in a
moderate temperature, produced when necessary by artificial heating, not
only is there very little, if any, restriction of the sexual season to a
particular time of the year, but that copulation in the winter is followed
by pregnancy. The following is a record of the cestrous periods (so far as
observed) and times of litters for one of Prof. Punnett’s rabbits from
October, 1910, to May, 1912 :—
434 Mr. Hammond and Dr. Marshall. Correlation between
Put to male. First notes urade on litter.
Sept. 27, 1910 Oct. 29, 1910
Jan. 26, 1911 Feb. 27, 1911
Mar. 29, 1911 Apr. 30, 1911
June 2, 1911 July 4, 1911
July 15,1911 Aug. 19, 1911
Sept. 2, 1911 ~ Oct. 6, 1911
Oct. 25, 1911 Nov. 27, 1911
Jan. 12, 1912 Feb. 15, 1912
Feb. 15, 1912 Mar. 17, 1912
Apr. 11, 1912 May 13, 1912
May 17, 1912 June 19, 1912
Heape states that 10-15 days is the average duration of the dicestrous
cycle, but that some individuals exhibit heat at intervals of three weeks.
The pro-cestrum is stated to last from one to four days, and cestrus for
about a day or longer. During the pro-cestrum the vulva tends to become
swollen and purple in colour, and this appearance may continue during
cestrus. There is no external bleeding, and it is difficult or impossible to
state when the pro-cestrum ends and cestrus begins. It would seem that
the two periods are much abbreviated, as in the case of the sheep and
many other animals in which the uterine changes characterising the heat
periods are slight, as compared with those of the dog or the monkey.
The uterus may show undoubted congestion at the heat period, but we
have never observed any breaking down of vessels or extravasation of blood
in the non-pregnant rabbit’s uterus, excepting near the end of the pseudo-
pregnant period. Itis possible that these (or some of these) cases represented
the commencement of a pro-cestrous period. Apart altogether from these
instances congestion presenting a close similarity to that observed in the case
of the pro-cestrous sheep was found to occur in the rabbit’s uterus at the
time of heat. Pigment formation has not been noticed. Its absence from
the uterus of the rabbit suggests that in this animal blood extravasation does
not ordinarily take place in the pro-cestrous or cestrous periods. The glands
do not show very much evidence of activity during the heat period, and their
degree of development is very much less than that shown in the earlier stages
of the pseudo-pregnant period.
Theoretical.
Many of the observations described above have an important bearing upon
certain statements made by Hill and O’Donoghue in a recent paper on the
cestrous cycle in the marsupial cat, Dasywrus viverrinus. According to these
authors ovulation in Dasywrus occurs at an interval of some days after
estrus, there being a definite post-cestrous period terminating in ovulation.
Ovaries, Uterus, and Mammary Glands in Rabbit. 435
Further, it is stated that the degenerative changes in the uterine mucosa of
the marsupial instead of preceding ovulation, as they do in the dog, take
place after ovulation during a period which, in the non-pregnant animal, is
designated the period of pseudo-pregnancy. The differences in the repro-
ductive cycles are shown in the following scheme drawn up by Hill and
O’Donoghue :—*
(1) Dasyvrvs. (2) EurHERIAN.
Aneestrus. Anestrus.
Pro-cestrus. Pro-cstrus (uterine degeneration).
|
Cstrus. Cstrus (ovulation).
cae
Post-cestrus (ovulation). Meteestrus.
Pregnancy. Pseudo-pregnancy Pregnancy.
(uterine degeneration).
Nursing period. Meteestrus. Nursing period. |
LK ’ ee
Ancestrus. Ancestrus.
Hill and O'Donoghue express the opinion that the degenerative changes
seen in Dasyurus during the pseudo-pregnant period are equivalent to those
which take place in the Eutherian during the pro-cestrum. They suggest
that the shortening of the cycle in the Eutherian may have induced an
increased growth of the mucosa during the pro-cestrum, and that this in time
may have conditioned the earlier recurrence of the degenerative and regenera-
tive changes, with the result that these have. been shifted forward so as to
oceur prior to ovulation instead of after it. On the other hand, Hill and
O’Donoghue appear to hold the view, which seems to us scarcely consistent
with the suggestion just quoted, that menstruation in man is a degeneration
of the uterine mucous membrane, due to its being unable to fulfil its purpose
owing to the absence of a fertilised ovum. They state, further, that their
observations “afford no support to the view that ‘menstruation is identical
with heat’ nor for the view that ‘menstruation in the Primates is the
physiological homologue of the pro-cestrum in the lower Mammalia.” Thus
they appear to regard the condition existing in the Primates as directly
comparable to that occurring in Dasyuwrus, and different from the condition
found in the dog.
Our own observations on the rabbit indicate that the changes in the non-
pregnant uterine mucosa which take place concurrently with the development
* Tn the scheme drawn up Hill and O'Donoghue “ Dicestrus” is unaccountably inserted
for the non-pregnant Eutherian between ‘ Pro-cestrus” and “ Metcestrus.” In the
scheme as given above, this is omitted, since the dicestrous period, when it occurs in
polycestrous animals, supervenes r metcestrum and not before.
436 Mr. Hammond and Dr. Marshall. Correlation between
of the corpora lutea are essentially similar to those described for Dasyurus
in the period of pseudo-pregnancy. The close likeness between the sections
of the rabbit’s uterus and the figures given in Hill and O’Donoghue’s paper
has been commented on above. Moreover, the processes which take place in
the ovaries and mammary glands are also clearly of an identical nature in
the two animals. We suggest, therefore, that the uterine changes which go
on in the pseudo-pregnant uterus in the marsupial are not comparable to the
pro-cestrous changes of the Eutherian, as Hill and O’Donoghue suppose, but
are identical with those in the pseudo-pregnant rabbit’s uterus, both being
dependent upon the formation of corpora lutea in the ovaries. It is possible,
however, that the uterine congestion occurring near the close of the pseudo-
pregnant period is of the nature of a pro-cestrous congestion, since pseudo-
pregnancy (like true pregnancy) would probably in some cases have been
followed by another cestrous period, had the animals been permitted to live.
It has been shown by Hill and O’Donoghue that in the marsupial cat there
is only one sort of corpus luteum, the duration of which is presumably always
the same. In the rabbit, also, there is only one kind of corpus luteum occurring
in correlation with either pregnancy or a condition comparable to pseudo-
pregnancy. The existence of only one kind of corpus luteum (which lasted
for an identical period, irrespectively of whether or not ovulation was suc-
ceeded by pregnancy) was no doubt the condition common to all primitive
mammals, and it seems probable that the shortening of the duration of the
“periodic corpus luteum ”* was associated with the development of the
polycestrous habit from a state of moncestrum. For it is known that
ovulation cannot ordinarily occur in the preseuce of fully developed corpora
lutea, which, if they persist, cause follicular atrophy and inhibit the develop-
ment of ripe ova. Consequently it would be disadvantageous for such animals
to have periodic corpora lutea persisting for as long a period as corpora lutea
associated with pregnancy.
In monestrous animals, such as the dog, the persistence of the corpus luteum
over a period equivalent to pregnancy would not be detrimental to fecundity,
while we have shown above that there is evidence (derived from numerous
cases where bitches have been known to secrete milk nine weeks after
cestrus) that even in the dog such a persistence may occur. Moreover, the
great variability which different individual dogs experience in the recurrence
of cestrus is suggestive of a variation in the period over which the corpus
luteum persists. It may be that in moncestrous animals the primitive con-
dition occurring in Dasyuwrus, in which there is one sort of corpus luteum
only, continues to exist or is reverted to in certain individuals.
* Or corpus luteum spuriun,
Ovaries, Uterus, and Mammary Glands in Rabbit. 487
Summary and Conclusions.
(1) The development of the corpus luteum of pregnancy, or of pseudo-
pregnancy, in the rabbit is functionally correlated with the hypertrophy of
the mammary glands, as already shown by Ancel and Bouin, and by
O'Donoghue.
(2) This hypertrophy is followed on about the 19th day after coition, in
pseudo-pregnant rabbits, by a definite secretion of milk, the quantity of
which may be temporarily augmented by the injection of pituitary extract,
just as in normal lactation. |
(3) The mammary hypertrophy can take place in rabbits from which
the uterus has been removed while still immature, thus showing that the
uterus is not an essential factor in the development of the mammary glands.
(4) The development of the corpora lutea of pseudo-pregnancy is further
correlated with uterine hypertrophy and hyperemia followed by extravasation
of blood. ni
(5) These uterine changes are clearly comparable to those which occur in
true pregnancy, and afford a confirmation of the view that the corpora lutea
are a necessary factor in causing and maintaining the raised nutrition of the
uterus during the first part of the period of gestation.
(6) The changes which take place in the rabbit’s uterus during pseudo-
pregnancy are homologous with those which occur in the uterus of the
marsupial cat during pseudo-pregnancy, and these latter are not pro-cestrous
in character (at any rate, in the earlier stages) as Hill and O'Donoghue suppose.
(7) The domesticated rabbit is capable of breeding throughout the whole
year, but less frequently in winter than in spring or summer. If corpora
lutea of pseudo-pregnancy are produced, the recurrence of cestrus is postponed
until these are in an advanced stage of retrogression.
(8) The shortening of the duration of the so-called corpus luteum spurium
of many mammals has probably been brought about in correlation with the
acquirement of the polycestrous condition.
The injections referred to in this paper were done by J. Hammond; the
operations by F. H. A. Marshall. The work was carried out at the Field
Laboratories, Cambridge, in connection with the School] of Agriculture. The
expenses have been defrayed by a grant made by the Board of Agriculture
and Fisheries out of money allotted to it, for purposes of research, by the
Development Commissioners.
[Postscript, March 6, 1914.—In describing the results of hysterectomy we
omitted to mention that Foges found that the uterus was not a factor in the
pubertal growth of the mammary glands.
438 Mr. Hammond and Dr. Marshall. Correlation between
Aschner and Grigoriu in a recent paper describe the effects of injecting
placental extract into virgin guinea-pigs. Development of the glands
followed, and this was succeeded by milk secretion. In the guinea-pig
ovulation may take place spontaneously, so that it is probable that there
was some gland development before the injections were made. Ovarian or
placental extract was found to cause hypereemia and other changes in the
uterus.
Fellner has lately described marked changes in the uterus and mammary
glands of the rabbit after injecting extracts of corpus luteum and placenta.
The organs affected are said to have undergone a considerable hypertrophy,
but milk production could not be induced.
Steinach has recorded experiments on guinea-pigs in which the ovaries of
females were transplanted into males and produced breast development.
Doncaster in a very recent paper on sterility in cats records a case of
what may be regarded as milk production following upon pseudo-preenancy.
Longley had previously observed that the cat, like the rabbit, normally
ovulates only after coition. One of Mr. Doneaster’s cats after copulating
with a tortoise-shell male failed to become pregnant. It occurred, however,
to one of the present writers that since copulation had taken place it was
probable that corpora lutea had been formed though unaccompanied by
pregnancy. It seemed possible, therefore, in the light of our experiences
with rabbits that the cat in question might secrete milk. This was found
to be the case four weeks after the last copulation, and Doncaster records
that the secretion continued for about two weeks subsequently. |
REFERENCES TO LITERATURE.
Ancel and Bouin, “Sur les Homologies et la Signification des Glandes 4 Secrétion
interne de l’Ovaire,” ‘Compt. Rend. Soe. Biol.,’ vol. 67 (1909).
Ancel and Bouin, “ Sur les Fonctions du Corps Jaune Gestatif. I—Sur le detérminisme
de la préparation de Vuterus 4 la fixation de lceuf,” ‘Journ. Physiol. et Pathol.
Générale,’ vol. 12 (1910).
Ancel and Bouin, “Sur les Fonctions du Corps Jaune Gestatif. II.—Sur le déterminisme
du développement de la glande mammaire au cours de la gestation,” ‘Journ.
Physiol. et Pathol. Générale,’ vol. 13 (1911).
Ancel and Bouin, “Sur PEvolution de la Glande Mammaire pendant la Gestation—
Déterminisme de la phase glandulaire gravidique,” ‘Compt. Rend. Soe. Biol,’ vol. 72
(1912).
Aschner and Grigoriu, “Placenta, Fotus und Keimdriise in ihrer Wirkung auf die
Milchdriisen,” ‘ Archiv f. Gynakol.’ vol. 94 (1911).
Basch, “ Uber experimentelle Auslisung der Milchabsonderung,” ‘ Monatsschr. f. Kinder-
heilk., vol. 8 (1909).
Biedl, ‘ Innere Sekretion,’ 2nd edition, vol. 2, Wien, 1913.
Ovaries, Uterus, and Mammary Glands in Rabbit. 439
Biedl and Koenigstein, “Ueber das Mammahormon,” ‘Zeitschr. Exp. Pathol. u.
Therapie,’ vol. 8 (1910).
Carmichael and Marshall, ‘The Correlation of the Ovarian and Uterine Functions,”
“Roy. Soc. Proe.,’ B, vol. 79 (1907).
Doncaster, “A Possible Connection between Abnormal! Sex-limited Transmission and
Sterility,” ‘Camb. Phil. Soc. Proce.,’ vol. 17, Part IV (1913).
Dubreuil and Regaud, “Sur les Relations Fonctionnelles des Corps jaunes avec |’Uterus
non gravide,” ‘Compt. Rend. Soc. Biol.,’ vol. 72 (1909).
Fellner, “‘Experimentelle Untersuchungen tiber die Wirkung von Gewebsextrakten aus
der Plazenta und den weiblichen Sexualorganen auf das Genitale,” ‘ Archiv fiir
Gyniakol.,’ vol. 100 (1918).
Foa, “Sui Fattori che determinanto |’Accrescimento e la Funzione della Ghiandola
Mammaria,” ‘ Archivio di Fisiol.,’ vol. 5 (1908).
Foges, “ Zur physiologischen Beziehung zwischen Mamma und Genitale,” ‘ Centralblatt f.
Physiologie,’ vol. 19 (1905).
Fraenkel, “ Die Function des Corpus luteum,” ‘ Arch. f. Gynikol.,’ vol. 68 (1903).
Frank and Unger, “ An Experimental Study of the Causes which produce the Growth
of the Mammary Gland,” ‘ Arch. Internal Medicine,’ vol. 7 (1911).
Halban, “ Die innere Sekretion von Ovarium und Placenta und ihre Bedeutung fiir die
Function der Milchdriise,” ‘Arch. f. Gynikol.,’ vol. 75 (1905).
Heape, “ Ovulation and Degeneration of Ova in the Rabbit,” ‘Roy. Soc. Proc.,’ B, vol. 76
(1905).
Heape, “The Source of the Stimulus which causes the Development of the Mammary
Gland and the Secretion of Milk,” ‘ Proc. Physiol. Soc.’—‘ Journ. of Physiol.,’ vol. 34
(1906).
_ Hilland O'Donoghue, “ The Reproductive Cycle in the Marsupial Cat Dasyurus viverrinus,”
‘Quart. Journ. Microse. Sci.,’ vol. 59 (1913).
Lane-Claypon, “On the Origin and Life History of the Interstitial Cells of the Ovary of
the Rabbit,” ‘Roy. Soc. Proc.,’ B, vol. 77 (1905).
Lane-Claypon and Starling, “An Experimental Enquiry into the Factors which
determine the Growth and Activity of the Mammary Glands,” ‘ Roy. Soc. Proc.,’ B,
vol. 77 (1906).
Longley, “ Maturation of the Egg and Ovulation in the Domestic Cat,” ‘Amer. Journ.
Anat.,’ vol. 12 (1911).
Mackenzie and Marshall, “ On Ovariotomy in Sows with Observations on the Mammary
Glands and the Internal Genital Organs,’ ‘Journ. of Agricult. Sci,’ vol. 4
(1912).
Marshall, ‘ The Physiology of Reproduction,’ London, 1910.
Marshall and Jolly, “ Results of Removal and Transplantation of Ovaries,” ‘Trans. Roy.
Soc. Edin.,’ vol. 45 (1907).
Niskoubina, “ Recherches expérimentales sur la Fonction des Corps jaunes,” ‘Compt.
Rend. Soc. Biol.,’ vol. 66 (1909).
O'Donoghue, “The Growth-changes in the Mammary Apparatus of Dasyurus and the
Relation of the Corpora lutea thereto,” ‘Quart. Journ. Microsc. Sci.,’ vol. 57 (1911).
O'Donoghue, “The Artificial Production of Corpora lutea and their Relation to the
Mammary Glands,” ‘Proc. Physiol. Soc.’—‘ Journ. of Physiol.,’ vol. 46 (1913).
Ott and Scott, “The Galactogogue Action of the Thymus and Corpus luteum,” ‘Proc.
Soc. Exp. Biol. and Med.,’ vol. 8 (1910).
Paton, ‘The Nervous and Chemical Regulators of Metabolism,’ London, 1913.
Schafer and Mackenzie, “The Action of Animal Extracts on Milk Secretion,” ‘ Roy.
Soc. Proc.,’ B, vol. 84 (1912).
WO, WOOK OF ib
440 Correlation between Ovaries, etc., in Rabbit.
Seitz, “ Die Follikelatresie wahrend der Schwangerschaft,” ‘Arch. f. Gynakol., vol. 77
(1906).
Steinach, “ Willkiirliche Umwandlung von Sdugetiermainnchen in Tiere mit aus-
gepragt weiblichen Geschlechtscharakteren und weiblicher Psyche,” ‘ Pfliiger’s Arch.
Physiol.,’ vol. 144 (1912). :
DESCRIPTION OF PLATES.
Puate 17.
Fig. 1—Microphotograph of mammary tissue of virgin rabbit (Experiment 1, p. 423).
The mammary development is limited to a few ducts.
Fig. 2.—Microphotograph of mammary glands of virgin rabbit which had ovulated
spontaneously about 14 days previously (Experiment 11, p. 425). The glands
contained numerous alveoli.
Fig. 3.—Microphotograph of mammary glands of rabbit from which the uterus had been
removed while still a virgin. It was killed 12 days after copulation (Experi-
ment 23, p. 430). The glands showed a great development of alveoli.
Puate 18.
Fi
=
ag
. 4.—Section through portion of mammary gland of rabbit 24 days after sterile
coition (Experiment 13, p. 425). The alveoli contain milk. x 78.
Fig. 5.—Section through uterine mucosa of rabbit nine days after sterile coition
(Experiment 9, p. 432). The glands are very greatly developed. x 35.
g. 6.—Section through uterine mucosa of rabbit 24 days after sterile coition (Experi-
ment 13, p. 432). A large quantity of extravasated blood is present. The
glands are still somewhat enlarged. x 35.
Fi
_
Figs. 4-6 were drawn by Mr. Edwin Wilson, of Cambridge.
Hammond & Marshall. Roy. Soc. Proc. B, Vol. 87 Pl. 17.
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'ammond & Marshall :
44]
Oxidation of Thiosulphate by Certain Bacteria in Pure Culture.
By Wiutiam T. LocKeErt.
(Communicated by Prof. P. F. Frankland, F.R.S. Received February 14,—
Read March 26, 1914.)
In the course of investigations on the oxidation of thiosulphate on bacterial
sewage filters,* it was found that partially oxidised filtrates, still containing
appreciable quantities of thiosulphate, were slowly but completely oxidised by
simple aération in the presence of living organisms, practically no oxidation
taking place in the control experiments with corresponding solutions rendered
sterile by steaming.
Further investigations were undertaken with a view to the isolation of the
organism or organisms capable of bringing about this oxidation.
Accordingly, gelatine and agar plates were made from active filtrates from
time to time and in general about 100 organisms per cubic centimetre were
noted, which were mainly of the non-liquefying and chromogenic types.
Subcultures in peptone water and peptone broth of several of the pre-
dominating types were made and after a few days’ incubation added to
solutions of thiosulphate, which were then aérated under sterile conditions.
Many experiments were carried out in this manner without success.
Variations were introduced with regard to the age of the cultures and
the nature of the culture media, without effect, practically no oxidation of
the thiosulphate solutions taking place after several weeks’ aération.
Subsequently it was observed that a bacteriological slide made of a
loopful of an active filtrate showed proportionately a greater number of
organisms per cubic centimetre than was indicated by the gelatine and
agar plates of the same solutions. Further, the microscopic appearance of
these—consisting mainly of one particular type—was very different from
that of the organisms previously subcultured.
All attempts to grow the particular and characteristic organism on the
usual media, ¢.g. nutrient gelatine and nutrient agar, failed. Minor investi-
‘gations indicated that the organism was most active in neutral solutions
‘containing only small quantities of organic matter, whilst ammonium sulphate
was a decided stimulant,
Ultimately it was found on plating out very small quantities (¢.g. 0:001 c.c.)
of an active filtrate on a solid gelatine medium made without bouillon, but
* © Journ. Soc. Chem. Ind.,’ June 16, 1913, vol. 32, No. 11, p. 579.
2L 2
442 Mr. W. T. Lockett. Oxidation of
containing ammonium sulphate (0-1 per cent.) and sodium thiosulphate
(0-4 per cent.), that a great number of slow growing, circular, non-liquefying,
bluish-white colonies were obtained. ‘
Plates made with such a medium showed that active filtrates contained
100 to 1000 times more organisms per cubic centimetre than was shown on
gelatine and agar. In addition the microscopic appearance of the organism
was apparently identical with those previously noted in the slides of the
filtrates.
Streak cultures of the organism made on media of the same composition as
the above produced clearly defined, thin, bluish-white growths after 10 to
15 days’ incubation at 20° C.
Experiments were then made to ascertain how far this particular organism
was able to bring about the oxidation of thiosulphate. At first difficulties
arose with regard to the finding of a suitable liquid medium for the growth of
the organism ; eventually good growths were obtained by the use of a medium
of the following composition :—
1:0 grm. sodium thiosulphate, 0°5 germ. ammonium sulphate, 0°5 grm.
potassium biphosphate, 0°025 grm. sodium chloride, 0:01 grm. magnesium
sulphate, 2:0 grm. Rochelle salt, dissolved in 1000 c.c. distilled water.
To this solution it was found necessary to add sufficient acid (N H2SO,) to
reduce the alkalinity to methyl orange by approximately one-half, thereby
presumably liberating free tartaric acid. Before and after sterilisation clear
solutions were obtained of this mixture, which were alkaline to methyl
orange:
A suitable solid medium for the growth of the organism is also obtained by
the addition of gelatine (10 per cent.) to this solution.
In testing the oxidising power of the organism the procedure generally
adopted was as follows:—A pure streak culture was taken, and a small
quantity of the growth, attached to the end of a sterile platinum needle, was
introduced into 10-12 c.c. of the above sterile solution contained in a test-
tube, the usual bacteriological precautions being observed.
After a few days’ incubation at 20° C.a slight white, stringy growth was.
observed in the inoculated solutions. Later, after 14-21 days a distinct
turbidity was apparent, and the solutions on examination at this period were
found to be free from thiosulphate. Complete oxidation had taken place with
the formation of acid sulphate, the final solution being slightly acid to methyl
orange.
Uninoculated solutions showed no change after several weeks’ incubation.
A large number of experiments have been made on these lines with
complete success. Solutions inoculated directly from colonies found on
Thiosulphate by Certain Bacteria in Pure Culture. 448
ammonium sulphate gelatine plates were similarly oxidised, and other
experiments have been conducted which confirm the above results.
The following is a typical example of the chemical results obtained :—
Results in parts per 100,000.
| Oxygen absorbed in |
three minutes from |
. acid permanganate.
Reaction with
mercurous nitrate.
Inoculated solution after 21 days’ incubation 1-00 White ppt.
at 20° C. |
Solution of control experiment after 21 days’ 28 -80* Black ppt.
incubation at 20° C. |
* Equivalent to 83-8 parts Na,S.O; per 100,000.
That the thiosulphate is bacterially oxidised to sulphate and that the
change is not a simple decomposition due to the formation of acid by the
organism seems evident from the fact that (1) there is no deposition of free
sulphur, (2) the final solutions do not absorb appreciable amounts of oxygen
from acid permanganate, this excludes the presence of thionic acids.
The following are comparative results obtained with three solutions, to one
of which had been added before incubation 1 c.c. of normal sulphuric acid,
thereby making the solution decidedly acid to methyl orange :—
Results in parts per 100,000.
| fa, |
| Oxygen absorbed in | : F
| ‘lice minutes from | See USD Remarks.
3 mercurous nitrate. |
| acid permanganate. |
i | |
| |
1, Inoculated solution after 21 days’ 1-80 White ppt. | Slight |
} incubation at 20° C. | turbidity.
2. Solution of control after 21 days’ | 21 -00* Black ppt. | Clear |
| incubation at 20° C. | | solution,
| 8. Solution made decidedly acid to 14 60 Yellow ppt. Deposit of |
| methyl orange prior to 21 days’ | (thionie acids). sulphur.
| incubation at 20° C.
* Equivalent to 61 parts Na,8,0,; per 100,000.
The organism is apparently able to live in slightly acid solutions, although
prolonged contact with free acid appears seriously to impair its activity and
growth.
Further experiments are in progress relating to the niorphology and
classification of the organism, which appears to be one hitherto unknown,
and to its effect on other sulphur compounds, ¢.g. tetrathionate.
444 Mr. A. E. Everest.
My thanks are due to Prof. P. F. Frankland, Dr. G. J. Fowler, and Edward
Ardern, M.Sc., for the interest which they have taken in this work and to the
Rivers Committee of the Manchester Corporation for permission to publish
the results of this investigation carried out in the laboratory of their sewage
works at Davyhulme.
The Production of Anthocyanins and Anthocyanidins.
By Artuur Ernest Everest, M.Sc., Lecturer in Chemistry, University
College, Reading.
(Communes by Prof. F. Keeble, F.R.S. Received February 16,—Read
March 26, 1914.)
The idea that the anthocyan pigments are closely related to the flavone and
flavonol glucosides is by no means new. Attempts to solve the problem
of their relationship have come chiefly from botanists, and, as a result of
their researches, a number of hypotheses have sprung up around which
quite considerable controversy has been centred. c
Miss Wheldale* puts forward the suggestion that anthocyan pigments are
the oxidation products of colourless or faintly coloured chromogens; and
that these chromogens are products of hydrolysis of glucosides present in
the tissues of the plant (probably glucosides of flavone or flavonol deriva-
tives). The hydrolysis of the glucoside she considers as essential to the
production of the anthocyan pigment. She represents the changes taking
place by means of the following equations :—
Glucoside + water = chromogen + sugar.
Then— Oxidation of chromogen > anthocyan pigment.
If this hypothesis be accepted, then either the anthocyan so produced will
remain a non-glucoside, z.¢., it will be an anthocyanidin, or in the presence
of sugars the anthocyanidin first formed must unite with sugar to form
an anthocyanin (glucoside), Her more recent suggestion that in flavone
glucosides all the hydroxyl groups are substituted by sugar molecules,
hence partial hydrolysis could produce glucoside anthocyans,} has apparently
no foundation upon experimental evidence, most of the flavone and flavonol
glucosides containing one or two sugar residues only.
Now, in view of the fact that it has recently been shown that in no case
* ‘Camb. Phil. Soc. Proc.,’ vol. 15, p. 137 (1909) ; ‘Journ. Genetics,’ vol. 1, p. 133 (1911).
+ ‘Biochem. Journ.,’ vol. 7, p. 87 (1913).
The Production of Anthocyanins and Anthocyanidins. 445
could any trace of anthocyanidin be found in any of the plants examined,*
there remains but one way of explaining their absence if Miss Wheldale’s
hypothesis is to be retained; namely, by assuming that the rate of forma-
tion of anthocyanin (glucoside) from anthocyanidin and sugar is greater
than that of the production of anthocyanidin from chromogen by oxidation,
and that these reactions take place under similar conditions.
If this were correct one would expect that by taking the yellow glucoside,
hydrolysing, then reducing without removal of sugars, the anthocyanidin
produced would combine with the sugar present to form an anthocyanin.
This is not the case. Evidence all tends to show that Miss Wheldale’s
view can no longer be accepted as explaining the available facts.
The reaction found so useful in determining the presence or absence of
elucosidal or non-glucosidal anthocyan, and already described by Willstatter
and Everest (doc. cit.), yields a very ready means of distinction between
these two classes of compounds, and has led to important results in the
present investigation. It depends upon the facts that anthocyanidins (non-
glucosides) are taken quantitatively from aqueous acid solutions, preferably
sulphuric acid, by shaking with amyl alcohol, whereas anthocyanins
(glucosides) remain quantitatively in the aqueous acid when similarly
treated; and, further, that the non-glucoside in amyl alcohol, shaken with
sodium acetate solution, remains quantitatively in the amyl alcohol, but on
shaking with sodium carbonate solution it is quantitatively carried down
into the aqueous layer.
The author has been able to show the production of anthocyanins from
yellow glucosides, and that in the cases where hydrolysed solutions were
taken only anthocyanidins were produced. That no intermediate formation
of anthocyanidins occurred when anthocyanins were obtained was shown
by carrying out the formation under amyl alcohol. Where glucoside
yellow pigments were used the anthocyanin appeared as usual, but no
anthocyanidin passed into the amyl alcohol; when a hydrolysed solution
was similarly treated the amyl! alcohol rapidly took up all the anthocyanidin
as it Was produced.
This makes it necessary to abandon the assumption suggested above as the
only explanation available if Miss Wheldale’s hypothesis is to remain.
' A number of papers have been published upon this subject by Keeble,
Armstrong, and Jones,+ and they conclude that the anthocyan pigments are
* Willstitter and Everest, ‘Annalen,’ vol. 401, p. 189 (1913).
+ ‘Roy. Soc. Proc., B, vol. 85, p. 214 (1912); B, vol. 86, pp. 308 and 318 (1918) ;
B, vol. 87, p. 113 (1913) ; and Keeble and Armstrong, ‘Journ. Genetics,’ vol. 2, part 3,
p. 277 (1913).
446 Mr. A. E. Everest.
produced in a manner similar to that propounded by Miss Wheldale, but
they part company with that author in regard to the process necessary
subsequent to hydrolysis of the glucosides, for they maintain that the
oxidation must be preceded by reduction of the non-glucoside flavyone or
flavonol derivative.
The result of work already published on the pigment of the cornflower,*
and consideration of the work of Keeble, Armstrong, and Jones, have led
the author to the conclusions (1) that if the anthocyans are produced from
the yellow glucosides, then it must be by some interaction in which the
glucosides and not the hydrolysed glucosides take part; and (2) that all
evidence obtained in dealing with the above-mentioned pigments tends
to show that the anthocyan pigments would prove to be, not oxidation,
but reduction products of the yellow glucosides.
That luteolin and morin give red pigments on reduction in acid alcoholic
solution by means of sodium amalgam has been known for many years.t
Quite recently Watsont has obtained a red pigment from quercetin by the
same means, and, lastly, since the present work was completed, the author’s
attention has been drawn to a paper by R. Combes,§ who describes the
production by the same means from the yellow pigment of the green leaves
of Ampelopsis hederacea of a pigment which he shows to be identical with
the red pigment (anthocyan) which he obtained from the red leaves of the
same plant. He does not, however, give definite information whether his
pigments are anthocyanins or anthocyanidins. The author is able to confirm
the production of anthocyan pigments by reduction of flavone or flavonol
derivatives, and to explain the observations of Keeble, Armstrong, and Jones.
Being one of the most commonly occurring of the flavonol class, and readily
obtained, quercitrin (Kahlbaum) was taken for the starting point.
Quercitrin by reduction with zinc (fine granulated) and dilute acids (2N HCl)
or by electrolytic reduction, even cold, gave only anthocyanidin.
The pigment production took place equally well when the aqueous acid
was covered with a layer of ligroin, so precluding all possibility of oxidation
by the air following reduction. As, however, no anthocyanin could be
obtained from quercitrin (this is a monoglucoside from bark, not a flower
pigment, at least some flowers are known to contain a diglucoside of
quercitrin, eg., viola contains viola quercitrin)—a result which at first
appeared to support Miss Wheldale’s hydrolysis hypothesis—the author
* Willstatter and Everest, loc. cit.
+ Cf. Rupe, ‘Die Chemie der natiirlichen Farbstoffe,’ vol. 1, pp. 77 and 85.
t ‘Chem. Soc. Proc.,’ 1913, p. 349.
§ ‘Compt. Rend.,’ vol. 157, p. 1002 (1913) ; ‘Chem. Zentr.,’ 1914, p. 158.
The Production of Anthocyanins and Anthocyanidins. 447
decided to test the pigments obtained by direct extraction of various flowers,
in particular with a view to producing anthocyanins from the yellow
glucosides present in them.
Having already shown that oxidation after reduction was not necessary
for the production of anthocyan pigments—and this was confirmed in every
case where reduction under ligroin was carried out—particular attention was
given to proving that anthocyanins could be produced directly from yellow
glucosides without intermediate formation of anthocyanidins, and in this the
author was successful.
The yellow wallflower, yellow daffodil, white narcissus, yellow or white
tulip, white primula (obconica), yellow crocus, yellow jasmin, yellow primrose
{the presence of yellow pigments in the white flowers was shown by action
of dilute ammonia, when, the plant acids being neutralised, the yellow colour
appears), and even lemon peel, all yielded by reduction alone red pigments,
and, indeed, pigments which upon investigation proved to be in every case
an anthocyanin, no trace of anthocyanidin being produced when the reduc-
tions were carried out in the cold. No oxidation after reduction was
necessary for the production of the anthocyanin pigment, provided that in
one or two instances care was taken not to carry the reduction too far.
- Reduction was carried out by zinc (fine granulated) in ca. 2N aqueous acids,
and also by electrolysis in 2N sulphuric acid, using lead electrodes (lead
has been found to yield salts with anthocyanins, which, however, are decom-
posed by acids; lead salts have no harmful effect upon anthocyanins).
At first some difficulty was experienced in explaining the observations of
Keeble, Armstrong, and Jones* that in the case of yellow wallflower, yellow
daffodil, yellow crocus, cream polyanthus, and Chinese primrose oxidation
was necessary after reduction in order to obtain a red pigment. A ready
explanation was, however, forthcoming when the case of the yellow tulip
was examined, for here, when reduction was rapid, there appeared but a
transient pink, passing rapidly to a colourless solution, which, however, on
addition of hydrogen peroxide immediately developed a red colour. Slow
reduction, however, by zinc (very small quantities) and HCl or, much better,
slow electrical reduction gave readily the red pigment, and this proved to
be as in the other cases an anthocyanin. The red solution on stronger
reduction passed to a colourless one, from which the anthocyanin was again
produced by the addition of hydrogen peroxide.
It has been found that in each case excessive reduction produced to a
greater or less extent the above result, and this clearly explains the results
of Keeble, Armstrong, and Jones (Joe. cit.).
* ‘Roy. Soc. Proc.,’ B, vol. 87, p. 113 (1913).
ABC Mr. A. E. Everest.
On a previous occasion attempts to reduce cyanin (the pigment of the
cornflower*) to a colourless compound which could be re-oxidised to the
pigment had failed. Powdered zine and acetic acid were used, hot—the
pigment was decolorised, but the red colour was not reproduced on addition
of hydrogen peroxide. Despite this fact, the author, for comparison, treated
a small quantity of cyanin chloride in 2N hydrochloric acid with much finely
granulated zinc, so that vigorous evolution of hydrogen ensued. The reaction
was carried out in the cold, and, as in the cases mentioned above, decolorisation
rapidly set in, but on decanting the decolorised solution and adding hydrogen
peroxide the colour reappeared. The glucoside was not hydrolysed by this
process.
In every case, also with cyanin chloride, when treated with hydrogen
peroxide in the cold, the red acid solution of the anthocyanin passed to
a yellow, then became colourless. It would thus seem that the balance of
reducing powers present in an anthocyan-containing flower must be very finely
adjusted, for it appears necessary that the reducing body present should be
powerful enough to reduce as far as the anthocyanin stage, but not powerful
enough to take the pigment further to the colourless condition.
It has been placed beyond doubt that the change from yellow to red may
be accomplished by reduction alone, thus confirming the results of Combes,
and, still further, that the change from glucoside flavone or flavonol to
anthocyanin (glucoside) takes place quite readily without hydrolysis, and
that all hypotheses which require a hydrolysis of the glucoside before
formation of red pigments can, in the light of the evidence of Willstatter
and Everest, that the anthocyanidins do not exist in plants, and the further
evidence now brought forward, that flavone or flavonol glucosides readily
yield anthocyanins without intermediate formation of anthocyanidins, be
discarded as unnecessary.
Whether all the yellow glucosides of the flavone and flavonol series are
capable of producing corresponding anthocyanins remains to be proved by
future work. The author failed to observe such formation in the case of
Primula sinensis (Giant white), mimosa, and white hyacinth. (Whites tested
with ammonia gave yellow.)
Whether the red pigments described above should be considered as mere
hydroflavone derivatives as I, or as some such anhydro-compound of them, II,
remains to be proved, but the author considers that the evidence at present
available favours some such form as II, where the change has caused the
production of a quinonoid structure, as follows :—
* Willstitter and Everest, Joc. cit.
The Production of Anthocyanins and Anthocyanidins. 449
H Cl
OH OH
HO ( ‘ty eS ow HO Ges ou
rE OH 8 aaOE
\WANY ae Acid AWA ie
@Q) @ reduction. Oo Cc
Jet | Jal AN
Ovi
H
I.
Cl
ms. pee
UN = IN
bis ad ee iow Das
SON NN oe
©
ET |
H
ele
In this connection an examination of the properties of cyanin chloride and
cyanidin chloride* is of interest. Cyanidin chloride when heated for a
short time in dilute alcohol to ca. 80° becomes decolorised—the decolorised
substance has properties resembling those of a yellow flavonol pigment,
soluble in ether, colourless in acid solution, extracted from it by ether ;
yellow in alkaline solution, and alkalis withdraw it from its solution in ether.
The decolorised cyanidin chloride, however, on boiling with acids, returns to the
red form. It is possible that these changes may be represented by the change
from I to II above being a reversible reaction. The fact that a decolorised
solution of cyanidin chloride on concentration regains its colour also
harmonises with the above.
Quite similar properties are observed in the case of cyanin chloride, save
that heat is not required for decolorisation, nor for return of the red pigment
on acidification. Extraction of the decolorised solution with ether was not
tried by Willstétter and Everest, but alkalis on the decolorised solution
gave a yellow coloration. Most probably the general character of the groups
in the molecule would have their effect on the readiness with which this
change took place, and hence this decolorisation. Such a change might
perhaps explain the observation of Keeble, Armstrong, and Jones (Joc. cit.),
that in the case of polyanthus mere boiling with acid was sufficient to
produce the red pigment.
As he learns that recent developments in the work of Prof. Willstatter
and his collaborators have caused them to commence a series of investigations
dealing with the relation between the yellow pigments and the anthocyans
the author proposes to discontinue these investigations for the present.
* Willstitter and Everest, Joc. cit.
450 Mr. A. E. Everest.
Experimental.
Quercitrin.—Reduction carried out in 2N HCl by zine (fine granulated).
(1) Hot, yielded rapidly a red solution.
(2) Cold, gave red coloration but very slowly.
(3) Cold, aleoholic HCl and Na Hg: rapidly gave red pigment.
(4) Cold, electrolysis in 2N sulphuric acid, lead electrodes: very slow pro-
duction of red.
(5) The best method, however, of obtaining the red pigment from quercitrin
is by the action of magnesium (ribbon or turnings) on a solution of the
substance in a mixture of 5 vols. absolute alcohol and 1 vol. concentrated
hydrochloric acid. Not only does this go very readily, but the acidity of the
solution—so essential in working with these compounds—is preserved as the
magnesium practically ceases to react before the solution becomes neutral.
This method was of no value when working with crude plant extracts, as
alcoholic extracts contained so much extraneous matter that the results were
masked.
In every case pigment, when shaken with amy] alcohol, went quantitatively
into the alcohol, solution red with tinge of violet; shaken with sodium acetate
solution, pigment remained quantitatively in alcohol, turned violet ; shaken
with sodium carbonate solution, pigment descended quantitatively into
aqueous layer with green colour; prepared by method (5) and purified from
remaining quercitrin, solution gave blue solution in sodium carbonate.
The red pigment was not extracted from aqueous acid by any other
organic solvent.
Yellow Wallflower.—Petals from a few flowers crushed in mortar with fine
sand and cold 2N HCl, then filtered, gave a faintly yellow extract which with
ammonia became deep yellow. To one portion of acid extract a small
quantity of zinc was added, whilst a second portion was kept for comparison,
to show that no red developed without the reduction. In a few minutes the
portion containing zinc became pink and the colour rapidly deepened to red.
Blank portion remained unchanged. The pigment produced by reduction
remained quantitatively in the aqueous layer when shaken with amyl alcohol,
but if the layer was separated, then boiled to hydrolyse the pigment, and then
again shaken with amyl alcohol, the red pigment then went quantitatively
into the alcoholic layer with production of a red solution. This reacted in
every way as an anthocyanidin. Electrolysis also produced the anthocyanin
and only that, no trace of anthocyanidin was produced by reduction in the
cold.
Reduction in hot solution produced anthocyanidin and no anthocyanin.
The Production of Anthocyanins and Anthocyanidins. 451
Reduction under ligroin produced the same results as without protection
from air.
Primula obconica (white)—Petals gave clear yellow on treatment with
ammonia, no pink with acids. Extract made as above, almost colourless.
Reduction with zine in cold 2N HCl gave good red pigment. The reduction
went equally well under ligroin, and in both cases the pigment produced was
quantitatively anthocyanin, and could be hydrolysed quantitatively into
anthocyanidin.
Primula sinensis (Giant white).—Petals gave clear yellow with ammonia,
no pink with acids. All attempts to obtain a red pigment failed.
Tulip (yellow).—Extract prepared as above in 2N HCl. With much
zine a faint passing pink colour appears, then solution becomes decolorised,
hydrogen peroxide added to the decanted solution causes appearance of red
colour. Exposure to air has same effect. The red produced is an anthocyanin
readily hydrolysable to an anthocyanidin.
When acid extract was treated with small quantities of zinc the pink colour
soon appeared and deepened. If not taken too far hydrogen peroxide caused
no change.
Electrolysis of cold extract in 2N sulphuric acid, lead electrodes, readily
gave the red pigment which, as in the preceding cases, proved to be entirely
anthocyanin.
Tulip (white).—Petals with ammonia gave clear yellow. Exactly similar
results were obtained as for the above yellow tulip. Both in the case of
yellow and white tulips the reduction went on equally well under
ligroin.
When the extract from the white tulip was boiled to hydrolyse the
glucoside contained, then cooled and reduced in the cold, a red .pigment
was readily obtained, but it was entirely an anthocyanidin.
Daffodil (yellow).—Extract as before, zinc in 2N HCl gave red pigment
easily. Electrolysis in 2N sulphuric acid gave same result. In both cases
cold reduction gave only an anthocyanin. Reduction went on to red pigment
equally well under ligroin.
Narcissus (small white).—Petals with ammonia gave clear yellow. Reduc-
tion with zinc in 2N HCl gave only anthocyanin.
Mimosa.—All attempts to get red pigment failed.
Hyacinth (white)—Petals gave yellow with ammonia, but all attempts to
obtain red pigment failed.
Crocus (yellow).—Extract as above gave, by zinc in 2N HCl, or by electro-
lysis in 2N sulphuric acid, red pigment quite readily, in both cases cold
reduction yielded only anthocyanin.
452 Mr. A. J. Walton. Growth of Adult Mammalian
Jasmin (yellow).—Gave anthocyanhingonly, more readily by means of zinc
and 2N HCl than by electrolysis.
Primrose (yellow).— Easily produced anthocyanin by either method, even
in fairly warm HCl with zinc only anthocyanin was produced. The glucoside
produced in this case seemed to be more stable to hydrolysis than in the
majority of cases.
Lemon Peel.—Extract in HCl, in presence of the peel, reduced with zine
gave only anthocyanin.
Variations in the Growth of Adult Mammalian Tissue in Auto-
genous and Homogenous Plasma.*
By Apert J. Watton, MS., F.R.C.S., B.Sc.
(Communicated by Prof. W. Bulloch, F.R.S. Received February 18,—
Read March 26, 1914.)
(From the Bacteriological Laboratory of the London Hospital.)
[PuatEs 19 anp 20.]
In 1910 Carrel commenced his researches on the growth of tissues outside
the body. In 1907 Harrison had succeeded in growing the embryonic tissues
of the frog, using coagulable lymph as a medium. In 1910 Harrison and
Burrows improved this method and successfully cultivated the tissues of
mammalian embryos. Carrel has so modified the technique that the
method is now applicable to the study of the growth of all mammalian
tissues. He used as a medium the plasma of the animal either in its
natural state or modified by the addition of various substances. Since
then, he and his collaborators have published a large number of papers,
and by their work it has been fully established that tissues of animals,
whether embryonic or adult, grow well im witro; that by changing the
medium and so removing the catabolic substances life can be greatly
prolonged—tissues have been kept alive and growing for periods con-
siderably longer than a year; and that the growth of the tissues can be
greatly modified by the addition of various substances to, or otherwise
altering the composition of, the plasmatic medium.
* Throughout this paper the term “autogenous” is used to indicate plasma obtained
from the same animal as the tissue, “homogenous” to indicate that obtained from
another animal of the same species.
Tissue n Autogenous and Homogenous Plasma. 453
In previous communications I have described fully the characters of
the growth of adult mammalian tissue in primary and in sub-cultures.
Having determined the nature of this growth, it became possible for me
to investigate the variation, if any, of the growth in autogenous and
homogenous plasma. The results of this investigation are detailed in this
paper.
The tissues of adult rabbits were used and the technique of Carrel was
carefully adhered to. Four hundred and thirty-three cultures were made,
the testicle, thyroid, and kidney being the tissues mainly used. A few
experiments were made with tissues of the spleen but owing to the
amount of emigration of cells, apart from true growth, which occurs with
this tissue it was found difficult to make accurate comparative experiments.
These experiments were therefore discontinued.
The details of the experiments on the testicular tissue and the results will
be fully described. The other tissues will be considered more briefly as the
experiments on them were carried out on the same lines.
Testicle.
One hundred and twenty-two cultures were made with testicular tissue.
It was found that the plasma of the rabbit kept in ice would not continue
fluid for longer than about an hour. After this time it coagulated and
became useless. To overcome this difficulty the plasma was placed in a
sterile tube which was corked and sealed with paraffin, and the tube was
immediately placed in a mixture of salt and ice contained in a thermos flask.
By changing the mixture of salt and ice every two or three days the plasma
could be kept frozen hard for an indefinite time. When required for use
it was removed from the mixture and at room temperature soon became fluid.
Experiment 1—Plasma was removed from Rabbit A six days previous to
the cultural experiment and kept frozen. At the time of the cultural
experiment blood was collected from the carotid artery of Rabbit B and the
plasma separated. Cultures of testicle of Rabbit B were made in the usual
way in both plasmata. Twelve cultures were made in the homogenous
plasma and twelve in the autogenous. Of those in the homogenous, good
growth occurred in all, whilst of those in the autogenous plasma only eight
grew and growth was less extensive in these.
Experiment 2—Cultures of testicle were made in homogenous plasma
which had been kept frozen for three days and in fresh autogenous plasma.
In both the same testicular tissue was planted and the cultures were made
at the same time and under identical conditions. The cultures in homogenous
plasma again gave 100 per cent. of positive results whilst those in autogenous
454 Mr. A. J. Walton. Growth of Adult Mammalian
plasma gave only 75 per cent. positive. The growth was again more extensive
in the homogenous plasma.
Although the above experiments seemed to show that growth was better
in homogenous than in autogenous plasma, it was possible that in both cases
the autogenous plasma happened to be what one may describe as a bad
variety. To solve this question a third experiment was performed three days
later than the second experiment. The same two plasmata which were used
in the second experiment had been preserved frozen and were used again.
They were now both homogenous to the culture tissue and were respectively
six and three days old.
Experiment 3.—The two frozen plasmata described above were used, one
of which was six days and the other three days old. Cultures of testicle
were made in these and in fresh autogenous plasma under similar conditions.
The results were somewhat similar to those of the last two experiments.
Growth was much more extensive in the homogenous than in the autogenous
plasma, thus at the end of three days there was but slight growth of the
tissue in the autogenous plasma and marked growth in the three-day-old
homogenous plasma, 100 per cent. of the pieces growing. In the case of the
six-day-old homogenous plasma it was seen that the growth was more
extensive than in the autogenous plasma but less than in the three-day-old
homogenous plasma, only 70 per cent. of the pieces growing. Sub-cultures
were made from each set and it was again seen that after a period of three
days growth was much more extensive in the homogenous than in: the
autogenous plasma. In the homogenous plasma mitotic figures were very
abundant and very well marked.
This experiment showed that growth was not better owing to the accidental
choice of good homogenous plasmata, for the plasma which in Experiment 2
was autogenous and gave but poor growth when used in Experiment 3, where
it was homogenous, gave a good growth in 100 per cent. of the trials.
Another very interesting fact became apparent. The homogenous plasma
used in Experiment 2 when three days old was successful in 100 per cent. of
the trials, but when used in Experiment 3, that is when six days old, gave
less growth, and even this appeared in only 70 per cent. of the tissues. These
results are shown tabulated on p. 455.
The facts suggested that the variations in growth might be dependent upon
the length of time that the plasma had been kept frozen, and further experi-
ments were therefore carried out to elucidate this point. No more sub-cultures
were made, however, for it was evident that if the same plasma were used
it would not be of the same age and therefore further variants would be
introduced.
Tissue in Autogenous and Homogenous Plasma. 455
Animal A. Animal B.
|
BIAS MB lA esesccs dase Homog., 3 days old. Homog., 6 days old. |
Good, 100 per cent. Medium, 70 per cent. |
Plasma 2 .....5...05+ Autog., fresh. | Homog., 3 days old.
_ Slight, 75 per cent. | Good, 100 per cent.
Plasma 3 ............ = : Autog., fresh.
Fair, 60 per cent. |
Experiment 4.—Plasmata were removed from two other animals and kept
frozen, one eleven and the other eight days before the cultural experiment.
Just before this experiment was commenced blood was removed from the
lateral ear vein of another animal and the plasma separated. Cultures were
then made under identical conditions in the four plasmata, viz.: homogenous
eleven days old, homogenous eight days old, homogenous fresh, and autogenous
fresh. In the first two groups every piece of tissue died and there was no
evidence of growth. The tissues in fresh plasma grew in the usual way and
to an equalextent. The growth in the autogenous plasma was perhaps a little
more extensive than that in the homogenous plasma (Plate 20, figs. 5 and 6).
These experiments showed that testicle grew better in homogenous plasma
that had been kept frozen for three days, but not at all in plasma that had
been frozen for more than six to eight days. The question as to whether
growth was better in autogenous or homogenous plasma was still undecided.
The following experiment was therefore devised to settle this point.
Experiment 5,—Two rabbits were taken. Blood was removed by puncture
from the lateral ear vein of each, ten and three days before the cultural
experiment, the plasma being separated and frozen. At the time of the
experiment blood was removed from the carotid artery of each and the testicle
taken out. Thus there were obtained from each animal three plasmata, one
which had been frozen for ten days, one for three days, and one fresh, that is
six in all. The testicle of each animal was cultivated in all the plasmata,
making twelve separate groups. The cultures were fixed at the end of
48 hours and stained so that the early growth-characters might be seen,
these being considered more capable of comparison than the later stages w ae
the growth was well advanced.
In the case of the testicle taken from animal A there was no trace of
growth in the ten-day-old plasma, whether taken from animal A or
animal B. With the three-day-old plasma that from animal A, autogenous,
showed well marked growth, but that from animal B, homogenous, showed
very slight growth and marked vacuolation of the plasma. With the
fresh plasma there was a fair amount of growth in both series, but whereas
VOL. LXXXVIL—B, 2M
456 Mr. A. J. Walton. Growth of Adult Manmalian
that in the autogenous plasma (fig. 4) was considerably less than that in
the three-day-old plasma, that in the homogenous was greater than that in
the three-day-old homogenous plasma and rather less than that in the fresh
autogenous. In the case of the testicle taken from animal B, there was
again no trace of growth in the plasma from either animal which had been
frozen for ten days (fig. 3), but in the three-day-old plasma there was
marked growth in the plasma from animal A, which in this case was
homogenous (fig. 2), and little or no growth in the plasma taken from
animal B, which in this case was autogenous. With the fresh plasma
there was growth in both series, but that in plasma A, homogenous, was more
marked (fig. 1) than that in plasma B but was much less than that in
the three-day-old homogenous plasma.
following table :—
These results are shown in the
Animal A. Animal B.
fr |
Plasma A. Plasma B. Plasma A. Plasma B. |
Autogenous. Homogenous. Homogenous. Autogenous. |
| |
4 x wo
1O(dayst eee 0) 0 0 0
6} CER AS godenuadoros Very good Slight Very good Slight |
Brel -2..ctreeemecs Good Fair Good Fair
The above experiments showed that, as regards the testicle, growth was not
dependent upon any variation in the nature of the cells, for growth was
equally good in the series whichever testicle was taken, but it varied directly
with the plasmatic medium which was used. The variations in the plasma
were not specific to either autogenous or homogenous tissues, for in the
experiments given above tissues from both animals grew in the one plasma
the other plasma
good growth and
evidence to show
whether it was autogenous or homogenous, whereas in
they grew badly in either case.
others but little, but at present there is not sufficient
upon what these differences depend.
The fact that growth was always better in plasma that had been frozen for
a certain time, whereas, if kept frozen for a longer period, growth entirely
ceased, seemed to show that each plasma contains two substances, one of
which inhibits growth and the other which stimulates it.
Some plasmata give
By exposure
to freezing for two or three days we may suppose the inhibitory substance
is destroyed so that growth is increased. After a longer period, about
eight days, the stimulating substance is also destroyed and hence there is
no growth. Under normal conditions the stimulating substance is in excess
Tissue in Autogenous and Homogenous Plasma. A57
of the inhibitory substance, therefore a certain amount of growth takes
place in fresh plasma. In plasmata which are not “good” only a small
amount of growth takes place when the plasma has been frozen for
three days. This is not so easy to understand; it may be that the
stimulating substance is present in a less marked degree, and is therefore
all destroyed at an earlier date, so that after the plasma has been frozen
for three days there will be little or none present, hence growth will be
slight or absent. It was noticed, however, in the cases above where growth
was slight that coagulation of the plasma had been incomplete; in some
cases, indeed, the plasma had remained quite liquid, so that there was
risk of the tissue washing off the slide. It is possible therefore that failure
to grow under such circumstances was due to mechanical factors, the
plasma failing to form a scaffolding for the growing cells. It is of interest
to note that the plasma which failed to coagulate was not serum, for there
was no clot present when the frozen material was thawed.
Thyroid.
Of this tissue 167 cultures were made, the experiments being carried out
on similar lines to those described for the testicle, but a larger number of
cultures were made, so that the plasmata were compared at shorter intervals
of time. :
Experiment 6.—Homogenous plasma was removed one day previous to the
cultural experiment and frozen. Autogenous plasma was removed from the
animal at the time of the experiment and cultures of thyroid tissue made
in each plasma under identical conditions. Growth was more marked in
the homogenous plasma and a greater number of cultures were positive in
this.
Experiment 7.—Thyroid tissue was cultivated in eight days’ old homo-
genous plasma and in fresh autogenous plasma. There was no growth in
the homogenous plasma, whereas in the fresh autogenous plasma 42 per
cent. of the cultures grew and the amount of growth was well marked.
Thus, as in the case of the testicle, growth is better in plasma that has
been preserved for one day, but entirely ceases in plasma which has been
frozen for eight days.
In the next series the same plasmata were used for several experiments, as
in the case of the testicle, so that any given plasma which was autogenous in
one experiment became homogenous in the next, and had been kept frozen
for periods of time which increased for each successive experiment.
Experiment 8.—Thyroid tissue was cultivated in fresh autogenous plasma
and in the plasma used in Experiment 7, which was now five days old.
2M 2
458 Mr. A. J. Walton. Growth of Adult Mammalian
The homogenous plasma gave 100 per cent. positive results and growth
was very well marked in it. Only 13 per cent. grew in the autogenous
plasma and growth in it was slight.
Experiment 9.—Thyroid tissue was cultivated—
(1) In plasma taken from animal 7, now nine days old.
(2) In the plasma taken from animal 8, now four days old.
(3) In fresh autogenous plasma.
In the nine-day plasma 60 per cent. of the cultures grew, the growth being
fairly extensive. ‘
In the four-day plasma 67 per cent. grew, and the growth was very well
marked.
In the fresh autogenous plasma 44 per cent. of the tissues grew, and growth
in these was less extensive than in the other groups.
Experiment 10.—Thyroid tissue was cultivated—
(1) In homogenous plasma taken from animal 7, now twelve days old.
(2) In that taken from animal 8, now seven days old.
(3) In that taken from animal 9, now three days old.
(4) In fresh autogenous plasma.
In the plasmata from animals 7 and 8 there was no growth at all. In the
plasma from animal 9 100 per cent. of the pieces grew and the growth of these
was very well marked. In the fresh autogenous plasma 44 per cent. grew.
The growth of these was much less marked than that of those grown in the
three-day homogenous plasma. ;
These results are shown in the following table :—
Plasma. Animal 7. Animal 8. Animal 9. Animal 10.
7 Fresh 5 days, 9 days. 12 days,
42 per cent. 100 per cent. 60 per cent. 0
8 —_ Fresh, 4 days, 7 days,
13 per cent. 67 per cent. 0
9 — — Fresh, 3 days,
44 per cent. 100 per cent.
10 = = = Fresh,
44 per cent.
It was clear that the increase in the amount of growth which took place
when the plasma had been kept frozen for about three days was very marked,
thus while autogenous plasma when fresh gave a growth in from 13 per cent.
to 40 per cent. of cases, it gave a growth in 100 per cent. of the trials when
it had been kept for three days and was homogenous. The fact that some
plasmata are good and others bad is also clearly shown by the’ table. For
instance, the plasma of animal 8 is definitely not so good as that of animals.
‘
Tissue in Autogenous and Homogenous Plasma. 459
J and 9. The results obtained in Experiment 9 are specially of interest, for
with plasma nine days old growth was obtained. This plasma coagulated well
but only gave 60 per cent. of positive results as compared with 100 per cent.
obtained with the same plasma when it was only five days old. This would
seem to show that the diminution of growth which occurred after the plasma
had been kept for a certain period was not entirely due to the lack of power
of coagulation, a lack which was considered the possible cause of failure in the
case of the testicular tissue. Further experiments were carried out to show
whether the increase of growth described above was due to the homogenity
of the plasma.
Experiment 11.—Thyroid tissue was cultivated in fresh autogenous and
homogenous plasmata. Cultures were also made in plasmata eleven and
eight days old respectively. As usual no growth took place in the last two
groups. In the fresh autogenous plasma growth occurred in 60 per cent. of
the cultures, whilst in the fresh homogenous plasma it was present in 40 per
cent. and was rather less marked.
The above results were confirmed by cross experiments carried out in the
same way as Experiment 5 was conducted in the case of the testicular tissue.
Experiment 12.—Blood was removed from the lateral ear veins of two
rabbits, eight and three days before the experiment. Fresh blood was
removed at the time of the experiment and the thyroids were taken out from
the two animals at the same time. Cultures were made from each thyroid in
all six plasmata. In the eight-day plasmata all four groups showed no
growth. The thyroid tissue of animal A showed positive results in 100 per
cent. of the trials in the three-day-old autogenous plasma, but no growth at
all in the plasma of animal B. With the fresh plasma again there was slight
growth in 59 per cent. of the cultures in the autogenous plasma of animal A,
and no growth in that of animal B. In the case of the thyroid of animal B
there was good growth in 100 per cent. of the trials in the three-day-old
plasma of animal A, which in this case was homogenous, and no growth in
that of animal B. With the fresh plasma there was fair growth in the plasma
of animal A in 64 per cent. of the cultures and none in the plasma
of animal B. These results are shown in the table on p. 460.
Thus this experiment confirmed what was found in the ease of the testicle,
namely, that growth was not dependent upon any quality of the cells or upon
the fact that the plasma was homogenous or autogenous, but one plasma was
bad so that neither tissue would grow in it, whilst the other was good and
gave good results.
460 Growth of Tissue in Autogenous and Homogenous Plasma.
|
Animal A. Animal B
2 ml
fs "ai
| Plasma A. § Plasma B. | Plasma A. | Plasma B.
Autogenous. Homogenous. | Autogenous. Homogenous.
8 days eee cece 0 0 | 0 0
DB CAB... 52. -de04e 20 100 per cent. 0 100 per cent. | 0
resin, scoot ete | 50 i () 64 es 0
| |
Kidney.
Of this tissue 96 cultures were made. The experiments were all carried
out by the method of cross growth, which made all the requisite points clear.
One such experiment may be quoted as an example.
Experiment 13.—Plasmata were collected in a manner similar to that used
in Experiments 5 and 12. The plasmata were respectively eight days old,
three days old, and fresh. In the eight-day-old plasma, as usual, no growth
took place. In the case of the three-day-old plasmata the kidney of
animal A grew well in the plasma of animal B but not at allin the plasma of
animal A. The kidney of animal B also grew well in the plasma of animal B
and not at all in the plasma of animal A. With the fresh plasmata growth
occurred in the case of both tissues in both plasmata, that in plasma B being
rather the better. Growth was in all cases less than that in the three-day-old
plasma of animal B.
The results of the kidney cultures supported therefore those obtained with
testicle and thyroid.
Summary.
1, The extent of growth of tissues in vitro is not dependent upon any
quality of the cells themselves.
2. The extent of growth varies directly with the character of the plasma.
3. The variation in the plasma does not depend upon whether it is
autogenous or homogenous but upon some cause at present unknown.
4. Fresh plasmata appear to contain substances, inhibitory and stimulating,
to the growth of cells, the latter being in excess.
5. The inhibitory substance is lessened, or the stimulating substance is
increased, by freezing the plasma for one to three days.
6. The stimulating substance is destroyed after the plasma has been frozen
for six to eight days.
Watton.
All x 35.
Roy. Soc. Proc. B, vol. 87, pl. 19.
Walton.
IV.
Vi.
oy. Soc. Proc. B, vol. 84, pl.
All x 35.
Iv.
20.
The Decomposition of Formates by B. colicommunis. 461
DESCRIPTION OF PLATES.
. Three days’ growth in fresh homogenous plasma.
. Three days’ growth in homogenous plasma three days old.
Three days’ growth in homogenous plasma ten days old.
. Three days’ growth in fresh autogenous plasma.
. Five days’ growth in fresh autogenous plasma.
. Five days’ growth in fresh homogenous plasma.
Dopp wd
The Decomposition of Formates by Bacillus coli communis.
By Ecerton CHARLES GREY, 1851 Exhibition Scholar.
(Communicated by Dr. A. Harden, F.R.S. Received February 19,—Read
March 26, 1914.)
(From the Biochemical Department of thé Lister Institute.)
Many observations have been made on the variability of gas production by
intestinal bacteria under natural conditions (see Penfold (1911) and Arkwright
(1913), where literature is quoted).
Penfold has found that by artificial selection of Bacillus coli communis in
the presence of sodium chloroacetate, strains may be isolated which produce
no gas from glucose and gas in lessened amount from mannitol, although in
both cases acid is produced as with the normal organism. The writer has
also shown that by artificial selection of B. coli communis by the chloro-
acetate method of Penfold, various stages between the original gas-producing
and the selected non-gas-producing strain may be obtained, and the changes
have been found to be associated in part with the disappearance of the enzyme
which decomposes formic acid (1914). It was found that two kinds of
artificially selected strains could be produced from the original strain of
LB. coli communis; one unable to decompose formic acid, and the other still
able to bring about this decomposition provided glucose were present.
The artificially selected organism, which could not decompose formates
even in the presence of glucose, was likewise unable to produce gas
from mannitol, whereas the organism which still retained the power of
decomposing formates was also able to produce gas from mannitol, although
it produced this gas in an amount approximately equal to one-half of that
produced under the same conditions by the original B. coli conumunis from
which it was derived. It seemed, therefore, likely that by a closer study of
462 Mr. E. C. Grey. The Decomposition of
the manner in which formic acid is decomposed by the natural and artificially
selected varieties of intestinal bacilli it might be possible to gain information
concerning the mechanism of the change brought about in the organism by
growth on chloroacetate agar which leads to the selection of strains in some
cases unable to decompose formic acid and in other cases unable to produce
it to the same extent as the normal strains from which they have been
derived.
It seemed also of importance to determine what use the decomposition of
formic acid might be to the organism. Pakes and Jollyman (1901) and
Harden (1901) have shown that B. coli communis is capable of decomposing a
considerable amount of sodium formate, and that if a small quantity of
glucose be added, the amount of hydrogen produced over and above that
which could be derived from the glucose added is far greater than the amount
produced in the absence of the sugar.
The writer has employed an artificially selected strain of 5. coli communis
obtained by the chloroacetate method ; this strain produced in three days no
gas from sodium formate peptone water, and only acid but no gas from
glucose peptone water, but produced from a mixture of the two sufficient
gas to fill the Durham gas tube (length 45 mm.) in 24 hours. The non-
production of gas from sodium formate peptone water alone is due, not to
the inability of the organism to decompose formic acid, but to the inhibitory
action of the alkali due to the natural alkalinity of sodium formate; for if
the sodium formate peptone water were acidified with sulphuric acid until
the solution imparted a pink colour to litmus, it was found that a small
quantity of gas was produced by growth of the artificially selected organism
therein for two or three days.
Other sugars and polyhydric alcohols have been employed with similar
results, which are discussed under Table II.
By a quantitative study of the decomposition by the bacillus in question
of a mixture of glucose and calcium formate, the writer has been able to
show that both the amount of glucose and that of formate decomposed is
increased (Table III), and there can be little doubt that the formate and
sugar are mutually helpful, in that the alkali produced by the decomposition
of the former and the acid produced from the latter by neutralising one
another maintain that approximately neutral condition of the medium which,
as has been proved, is most favourable for the action of the organism.
Formates by B. coli communis. 463
EXPERIMENTAL.
The Examination of the Behaviour of Non-gas-producing Organisms towards
Formates as a means of Deciding whether the Organism has been Derived
rom an Original Gas-producing Strain.”
It has been mentioned above that by artificial selection of B. coli communis
it is possible to obtain strains which do not produce gas from glucose,
and that this phenomenon consists in part, in some cases, in a lessened
power to decompose formic acid possessed by the selected organism.
In the case of the strains examined by Penfold and Harden (1912) the
power of decomposing formic acid was in all cases retained by the
selected strains, and certain strains examined in the course of this work
were found likewise to have retained this power. In the case of one strain,
however, the power to decompose formic acid had been entirely lost. It
may, therefore, be considered as probable that the strain incapable of
decomposing formic acid represents a more advanced stage in the process
of selection, and that, therefore, this type would be more permanent in
character. Such indeed has proved to be the case, for while the strain
which retains the power to decompose formic acid tends to revert in its
properties to the parent organism as regards the production of gas from
glucose, the other strain, which cannot decompose formic acid, shows no
such tendency, although it has been frequently sub-cultured during the course
of seven months.
In view of the fact that the more permanent non-gas-producing type of
artificially selected strain is unable to decompose formic acid, it may be
suggested that the same phenomenon might be exhibited by naturally
occurring non-gas-producing organisms, and that in order to decide whether
a strain which, at any particular time, does not produce gas has been
recently derived from a gas-producing strain, an examination of its behaviour
towards formic acid might be of crucial importance.
It frequently happens that organisms isolated from natural sources differ
apparently only as regards the power to produce gas from carbohydrates and
allied substances, and the question arises as to whether the one organism
may have recently been derived from the other. Arkwright (1913), for
example, has obtained varieties of B. acidi lactici differing in the aforesaid
respect, both strains occurring in the same sample of urine, and he was
also able to show that im certain cases the non-gas-producing strain could
be trained to decompose sodium formate if grown for some time on a peptone
water medium containing this salt. The writer has found that the power to
produce gas from mannitol may, in some instances, be made to disappear by
464 Mr. E. C. Grey. The Decomposition of
simply allowing a broth culture of B. coli communis to remain unchanged for
three months, or by growth of the gas-producing organism anaérobically in
peptone solution containing mannitol in the presence of chalk for about
one month. At the end of the period described, if a loopful of the culture
be plated out on to agar, many of the colonies which grow at 37° will be
found to produce no gas when inoculated into mannitol peptone water
tubes. This change may be seen from Table I.
Table I.—The Disappearance from B. coli communis of the Power to
Produce Gas from Mannitol by Continuous Growth of the Normal
Organism in Unchanged Cultures.
Production of gas.
History of the culture. a Sara
Mannitol. Glucose.
Normal B. coli recently isolated, average 46 normal 30 mm. gas 21-0 mm. gas.
strains
The above-mentioned normal strains after being kept 25 8 22 °0 »
in unchanged broth 4 months, average 6 strains
Kept in unchanged broth 4 months, average 12 strains 2am ee 20°0
»? »” 4 ” ” 12 ” 5 ” 18 ‘o ”
2» ” 4: » ” 8 By} 2 Bo) 20° ”
33 3) 4 2) > 9 39 Nil 21 ‘0 ”?
The strains described in Table I, which did not produce gas from mannitol,
were examined after growth on broth during several sub-cultures and were
found not to produce gas from mannitol when inoculated from the broth
tubes into mannitol peptone water. Thus the acquired character is inherited
for a considerable time under these conditions. It will be seen from the
foregoing table that no change has been brought about in the power to
produce gas from glucose, and this is also true for dulcitol. Nevertheless,
if by simple growth in peptone water B. coli communis yields a strain
incapable of producing gas from mannitol, it would seem not unlikely that
some similar process might, with time, lead to the disappearance of the
power to produce gas from glucose, but such has not so far been observed.
In deciding whether an organism possesses the formic acid decomposing
enzyme, which it is suggested here should be used as a criterion of a gas-
producing organism, it is not convenient or sufficient to observe whether gas
is produced from peptone water containing sodium formate. The test should
be made with a mixture of sodium formate and glucose in such proportions
that the sodium carbonate which will result from the decomposition of the
formate will be approximately sufficient to neutralise the acid which will
Formates by B. coli communis. 465
be produced from the carbohydrate. A convenient mixture is 1°5 per cent.
carbohydrate and 0-5 per cent. sodium formate in 1 per cent. peptone water.
It will be found under these circumstances that whereas an organism may
give only a few bubbles, or even no gas at-all, from sodium formate peptone
water alone, and none at all from glucose peptone water alone, the mixture
may yield gas with great rapidity, so that in 20 hours a Durham tube may
be completely filled. This increased gas production is due chiefly to the
decomposition of the formate, but partly also to gas which may be produced
from the sugar when the solution is maintained neutral, as will be described
later.
This increased gas production from formates in the presence of carbo-
hydrates is strikingly illustrated in the case of a selected strain of
B. coli communis obtained by the chloroacetate method, as will be seen from
the following table. The numbers represent millimetres of the tube
occupied by gas in the Durham tubes of 45 mm. length.
Table I1—The Effect of Addition of Carbohydrates and Allied Substances
on the Decomposition of Sodium Formate by an Artificially Selected
Strain of B. coli communis producing only a Minute Quantity of Gas
from Glucose.
| | |
| |
| 7 | | |
Time. | 5 ae Glucose. : Lactose. | Mannitol. Dulcitol. | Sorbitol. Glycerine.
| | |
(Concentration of the Sugar or Alcohol 2 per cent.)
hours | | |
12 Nil Nil Nil Trace Nil | ‘Trace Nil
24, 2»? » | ” 11 ” 12 ”
36 ms Minute | Minute 23 . 25 pp
bubble | bubble
60 5 No increase 0°5 No increase 5 No increase Trace
84 ; j en|aiieaco , Bid Uses
108 = 5 I > 630) * By | i 1
132 #5 | y 3°0 a 37 % 1
Evolution of Gas from the above Carbohydrates and Alcohols after
Admixture with Sodium Formate.
(Carbohydrate or Alcohol 1°5 per cent., Sodium Formate 0-5 per cent.)
1Zay,| 12 4:0 | Trace Nil 2 Nil
24, Ful | Full 10 3 10 | in
36 | 34 55 Full | 5
60 | | Full i 5
| 84, | 5 7
|
The following facts should be noted in connection with the experiment
described above :—
466 Mr. E. C. Grey. The Decomposition of
(1) The non-production of gas from formate peptone water alone was due,
in part, to the natural alkalinity of the medium. To demonstrate this
varying quantities of N/10 H2SO, were added to a series of sodium formate
peptone water tubes, which were then inoculated with a loopful of a broth
culture of B. coli communis. It was found that in those tubes in which the
reaction to litmus was nearest to neutral, there was a slight production of
gas, whereas those which were distinctly alkaline or acid showed no gas
at all.
(2) The manner in which the inoculation is made is also of importance.
Several tubes of sodium formate peptone water were inoculated each with a
loopful of a broth culture of B. coli, and another set of tubes were inoculated
each with a loopful of an agar growth of the same organism. The former
set of tubes produced no gas, the latter produced one-tenth of a Durham
tube. This difference in the production of gas cannot be due simply to the
size of the inoculation, for even when kept for 10 days the formate tubes
inoculated from the original broth culture showed no production of gas.
Probably, therefore, the bacillus when grown on agar contains more of the
formic acid decomposing ferment than when grown in broth.
(3) The decomposition of sodium formate is not assisted in the same
degree by mannitol as it is by glucose and the other sugars or by sorbitol,
and it may be possible that this phenomenon is related to the fact already
mentioned, that the power to produce gas from mannitol disappears from
old broth cultures of B. coli communis, when these have remained unchanged
for some months, and still more readily when the fluid contains mannitol
and the products therefrom.
It should be noted also that, since less acid is produced from a hexahydric
alcohol than from the same weight of a hexose when fermented by B. coli
communis, the fact that the alcohol does not assist so well in the acceleration
of the decomposition of the formate by the organism is in harmony with the
view that it is the neutralisation of the medium by the acid produced by
the carbohydrate or allied substance which is of assistance for the further
decomposition of the formate.
The fact that in any particular experiment no gas may be produced from
glucose peptone water is not a complete proof that an organism cannot
produce gas at all from glucose, for the acid produced under circumstances
in which no precaution is taken to neutralise the medium inhibits the
decomposition of formic acid.
Formates by B. coli communis. A67
Quantitative Study of the Rate and Extent of Decomposition of Sodium Formate
and Glucose by an Artificially Selected Non-gas-producing Strain of B. coli
communis when grown on them either separately or together.
In order to determine the causes of the greatly increased gas production
observed when B. coli communis was grown on a mixture of sodium formate
and glucose, the change was followed quantitatively. For this purpose it
was necessary to determine the weight of formic acid and glucose consumed
in the reaction and the total carbon dioxide and acid produced, and also to
measure the gas production from time to time. The method which was
employed would be suitable for the examination of the decomposition of
many other substances by bacteria, and it is therefore described in detail.
A quantity of 50 or 100 cc. of 2 per cent. glucose in 1 per cent. peptone
water is sterilised and inoculated with the organism. The cotton-wool plug,
which should fit loosely, is pushed half-way down the neck of the flask, and
the flask is connected with a Schiff’s gas burette by means of a rubber
stopper provided with a two-way tap. The burette, which is filled with
mercury, is in connection with a reservoir for adjusting the pressure, as in
_ the apparatus described by Harden, Thompson, and Young (1910). Before
beginning the experiment, air may be removed from the flask by putting it
in connection with the burette. On lowering the reservoir air passes into
the burette. Nitrogen is then admitted to the flask by reversing the tap,
and this process is repeated four or five times, when the oxygen will have
been practically all removed. The flask is well immersed in a water-bath
maintained at 37°. When it is desired to stop the reaction, the flask is
removed from the water, and the contents are, after turning the two-way
tap so as to put the flask in connection with the apparatus described below,
carefully brought to the boii, the gas driven out displacing the mercury
from the inner tube A (see figure).
Detwils of the Use of the Gas Collecting Apparatus.—The object of the
apparatus is to collect all the gases which remain in the fermentation flask
both free above the surface of the medium and dissolved in the fluid.
A is an ordinary Liebig’s condenser set vertically and connected by a
three-way tap D with a gas burette B accurately graduated. By putting D
in connection with the pump or by raising the tube C, which must be filled
with mercury, the mercury rises to fill B; the tap E is then closed. The
tap D may now be reversed and mercury drawn up into the inner tube A
from the reservoir L’” to the level L’. A circulation of water in the Liebig’s
condenser is not necessary for the condensation of the steam, but helps in
keeping the temperature of the collected gas constant. To collect the gases
468 Mr. E. C. Grey. The Decomposition of
the flask F is heated carefully and the contents brought to the boil; the gas
displaces the mercury from the inner tube A, and should the gas evolved
be more than sufficient to fill A the tap D may be turned so as to connect
A and B, and the tap E turned so as to connect B and OC, while C is lowered ;
the mercury in B falls with a corresponding rise of mercury in A.
The volume of the inner tube from a definite etched mark L’’ to the tap
Formates by B. coli communis. 469
D, including the volume of the pressure tubing connecting A and B, having
been previously determined, the total volume of evolved gases may be
measured by raising the reservoir C, E being open, and D turned to connect
A and B; the mercury then rises in B and-falls in A, in which it is allowed
to fall to the level L’’. To correct for pressure an allowance may be made
for the height of the mercury from the surface of the reservoir L’”’ to L”,
but it is also quite convenient to lower the whole Liebig’s condenser until
L” coincides with L’’. The volume of gases in the graduated tube B is
then observed, and this volume added to that of the inner tube A. A sample
of the gases may now be conveniently removed by lowering C. When B
contains sufficient of the gases for analysis, the whole apparatus B-C may,
if desired, be removed from its connection with A.
The apparatus has been described in detail because it is of use for the
determination of all gases remaining in the fermentation flask. In the
experiments recorded in the present communication, however, it was only of
value to determine residual carbon dioxide.
Details of the Determinations.—The carbon dioxide boiled off from the
solution, as described above, is measured in the usual way. The flask is
now detached from the apparatus and the contents filtered from the deposit
of chalk, and the filtrate and washings precipitated in hot solution with
ammonium oxalate. The precipitate of calcium oxalate is used to estimate the
calcium corresponding to the total acids produced during the fermentation,
an allowance being made for the calcium in the peptone. ‘The filtrate from
the calcium oxalate is acidified with oxalic acid and distilled in steam, the
distillate neutralised with deci-normal potash and evaporated to dryness ;
the residue is dissolved in about 50 ec. of water, and the formic acid
determined by the reduction of mercuric chloride. The residue from the
steam distillation is made up to a definite volume, and an aliquot portion
used for the determination of the residual sugar by Bertrand’s method after
the removal of peptone by Patein’s mercuric nitrate reagent.
The results of the experiment are summarised in Table III.
It will be seen from .Table III that about ten times as much gas was
produced by the selected strain of B. coli communis from calcium formate in
the presence of glucose as was produced by it from calcium formate alone.
The amount of sugar decomposed in the presence of calcium formate is
considerably greater than in its absence, even when the medium is kept as
far as possible neutral by chalk.
470
Mr. E. C. Grey. The Decomposition of
Table II1I—Comparison of the Action of an Artificially Selected Strain of
L. coli communis (Escherich) on Glucose alone; Glucose + Calcium
Formate ; Calcium Formate alone.
Conditions of the experiment.
Glucose alone. | Glucose alone.
Medium not Medium kept | Glucose + calcium | Calcium formate
neutralised during| neutral by formate + chalk. alone.
fermentation. | chalk.
Duration. esse eee 99 hours 99 hours 120 hours 120 hours
Glucose before 3 °385 1 -6926 1 °6926 None
jn aftercare: 3 2276 1 -0628 None 4
a consumed 0°1574 0 -6098 1 °6926 -
Formic acid before None None 0 °5244, 0 5244
» yy after 00874 0 0249 0 0276 0 :4988
on) yeep te = = 0 -4968 00256
sumed.
CO, total gas ...... 42 cc. 96 c.c. 291 c.c 12 c.c.
CO, from acids on 41 ,, SO GI, -
chalk
CO, from formate _ — init)" 12 ¢.c.
CO, from sugar ... - 6 cc. DLs -
The medium contained in all cases 1 grm. peptone (Witte) in 100 c.e.
The actual carbon dioxide produced by the organism from calcium formate
is in reality twice that actually evolved, for in the decomposition
(HCOO).Ca + H20 = CaCO3+CO2+ 2H:
it is clear that one-half of the CO2 is retained in combination with the
calcium. .
These results bring out, therefore, very clearly one object which is attained
by the decomposition of formates by these bacteria, viz.: that the organisms
are thereby supplied with the best possible neutralising agent. For the
formate by being decomposed into carbon dioxide and hydrogen virtually
liberates alkali within the bacterial cytoplasm, and thus not only neutralises
the medium, but also the bacteria themselves. Moreover the calcium formate
being itself neutral possesses none of the disadvantages which would arise
from the presence of even a slight excess of alkali. It would be difficult to
devise a more efficient means for maintaining neutrality in this case. I
would suggest the utilisation of sodium or calcium formate as a neutralising
agent in working with those organisms capable of decomposing it, especially
for solid media, with which the addition of dissolved alkali from time to time
would be impracticable.
Formates by B. coli communis. 471
Summary and Conclusion.
(1) The power of B. coli communis to decompose formic acid varies con-
siderably when the organism has been kept for some time on artificial
media.
(2) The decomposition of formates is inhibited by a very small excess of
either acid or alkali and, therefore, a greatly increased decomposition of
formates results if glucose is added, since the acid produced from the sugar
neutralises the alkali from the formate.
(3) A method and apparatus are described by which the decomposition of
various substances by micro-organisms may be followed quantitatively
requiring only 50-100 c.c. of the solution.
(4) It has been suggested that in place of a solution of sodium formate a
mixture of sodium formate 0°5 per cent. and glucose 1:5 per cent. should be
used as a test of a gas-producing strain, since by this means the production
of gas from formate is greatly increased, and it is also suggested that the test
could be used as a criterion as to whether an organism, which has been
recently isolated from some natural source and produces no gas from glucose
peptone water, may be regarded as having been recently derived from a gas-
producing strain.
(5) It has been shown that formates may be conveniently used as
neutralising agents, and that thereby the activity of gas-forming organisms
may be considerably increased.
In conclusion I would express my thanks to Prof. Harden, F.R.S., for help
and criticism.
REFERENCES.
Arkwright, J. A., ‘Journ. Hyg.,’ vol. 13, p. 68 (1913).
Grey, E. C., this vol., p. 472 (1914).
Harden, A., ‘Chem. Soc. Journ.,’ vol. 79, p. 610 (1901).
Harden, Thompson and Young, ‘ Biochem. Journ..,’ vol. 5, p. 230 (1910).
Harden and Penfold, W. J., ‘Proc. Roy. Soc.,’ B, vol. 85, p. 416 (1912).
Penfold, W. J., ‘ Roy. Soc. Med. Proc.,’ p. 97 (1911).
Pakes and Jollyman, ‘Chem. Soc. Journ.,’ vol. 79, p. 386 (1901).
WO, WxODAVN sy 2N
472
The Enzymes which are Concerned in the Decomposition of Glucose
and Mannitol by Bacillus coli communis.
By EcGerton CHARLES GREY, 1851 Exhibition Scholar.
(Communicated by Dr. A. Harden, F.R.S. Received February 19,—Read
March 26, 1914.)
(From the Biochemical Department of the Lister Institute.)
By the cultivation of bacteria in the presence of certain substances, for the
most part toxic in character, it is possible to obtain strains in which the
fermentative powers differ considerably from those of the parent organisms.
As an example may be taken a variety of B. coli communis (Escherich) which
was produced by the growth of that organism on agar containing sodium
chloroacetate (see Penfold, 1911). This strain differed from the parent strain
in that it now decomposed glucose with the production of acid but not of gas.
This result pointed to two possibilities ; firstly the decomposition of glucose
by the selected strain might be brought about by a set of ferments, which
acted very differently from those of the normal strain responsible for the
decomposition of the same substance, or secondly the primary cleavage
products of glucose might be the same both from the original and the
selected strain, and the difference between the action of the two might
depend upon some secondary process, as for example the decomposition of
formic acid, through which, as Pakes and Jollyman (1901) and Harden
(1901) have shown, the carbon dioxide and hydrogen most probably arise.
It is obviously of great biological importance to know whether the changes
brought about by growth on sodium chloroacetate result in any profound
modification in the carbohydrate metabolism of the organism. It was, at the
outset, considered most probable that those enzymes which were responsible
for the cleavage of the glucose molecule into its primary products would be
less likely to be lost than those which brought about the secondary changes.
It was hoped, therefore, that by a comparison of the products formed from
glucose and mannitol by the normal organism with those produced from the
same substances by the artificially modified strains it would be possible to
determine how many different enzymes were concerned in the process.
Ifa number of products are formed by one enzyme the ratio which they
bear to one another should not be altered by the process of selection, or
conversely if on selection the ratio between any two substances is found to
alter it may be taken as evidence that these two substances are not produced
Decomposition of Glucose and Mannitol by B. coli communis. 473
by one enzyme, unless these two substances can replace one another to a
certain extent owing to secondary reactions.
Isolation of the Organism.
B. coli communis (Escherich) was chosen for this work since the first
observations made by Penfold (1911) on the disappearance of the gas-
producing power by growth in the presence of sodium chloroacetate were
made with this organism. It was found, however, that very many strains
ot B. coli communis could be isolated, showing not only differences in degree
(which need not be considered here), but also of kind.
The organisms were isolated from human feces in the ordinary way. A
broth culture was made and from this bile salt cane-sugar neutral red agar
plates were inoculated; after incubation for one or two days at 37°, a
number of white colonies (cane-sugar non-fermenters) were removed to tubes
containing lactose peptone water coloured with litmus, and provided with
Durham gas tubes. Those tubes which on incubation produced acid and gas
(lactose fermenters) were used to inoculate a series of tubes containing
dulcitol peptone water. By these three operations organisms were obtained
which according to MacConkey (1905) belonged to the B. coli communis
(Escherich) group. The general characteristics of the four varieties which
were found will be seen by reference to Table I.
Table I.—Characters of Strains of B. coli communis occurring together in
Normal Feces.
| Fermentation of sugars, etc. |
|
i |
|
ae | Indole pro-| Milk
| ae diichion. | clotting. | |
| | | Glucose. | Lactose. | Mannitol. | Cane-sugar. |
1. Rapid ...... Strong 24 hrs A,G@ A,G | IN (Ge Nil |
2. Slight ...... Medium | 24 ,, ep eauG: A,G A,G Nil
3. Slight ...... | Strong | Bidays liq AGG, ARS: | A,G@ Nil |
4, Rapid ...... Ni | 24hbrs | A,G A,@ | A,G Nil) he |
|
All the above strains were Gram negative, did not liquefy gelatin and did
not give the Voges and Proskauer reaction. Very many examinations were
made of the motility in from 3 to 10 hours’ cultures.
The most striking difference is that between the rapidly motile
No. 1 and the practically non-motile No. 2. These were chosen, therefore,
for further study, since it seemed possible that the motile organism might
differ considerably in its metabolism from that which was slightly motile.
2N 2
474 Mr. E. C. Grey. Decomposition of
The consideration of this relationship is, however, not one of the objects of
the present communication.
It is important to note that the difference in motility between strain
No. 1 and No. 2 is not merely one of degree but rather one of kind. It is,
as a matter of fact, rather difficult to decide whether No. 2 is really motile
at all, and only after concentrating the attention on one bacillus and
observing its position from time to time in relation to an adjacent organism
is it possible to decide that it really does exhibit a motion of translation.
The strain was examined very many times in cultures from 3 to 12 hours’
growth and at later periods, but no increase in the motility of this strain
was ever observed. With the strain No. 1 the appearance is entirely
different ; in cultures of any age from 3 to 24 hours, rapid motility is readily
observed. In cultures less than 9 or 10 hours’ old the bacilli may be seen
travelling with such rapidity that it is almost impossible to follow the course
of any one particular bacillus. In young cultures (3 to 7 hours) the bacilli
may be readily seen in long threads, in which the bacilli have not had time
to separate. No such threads were obtained with strain No. 2.
The highly motile typical B. coli communis (Escherich) will be referred to
as No. CI, and the feebly motile strain as No. CF.
Artificial Selection of Non-gas-producing Strains by Growth of B. coli communis
(Escherich) on Agar containing Sodium Chloroacetate.
The technique of the chloroacetate method of selection has been described
by Penfold (1911) and has been closely followed here. It has been found,
however, that there is very considerable variation in the power of resistance
to sodium chloroacetate, and also in the appearance of the chloroacetate agar
plates inoculated with various strains of B. coli (Escherich). The nature of
the changes brought about by growth in the presence of sodium chloro-
acetate will be discussed in a separate communication, and it must suffice to
say here that the changes do not merely consist in the simple disappearance
of the power to produce gas from glucose, but are, rather, of such a nature
as to affect, to a greater or less extent, most of the enzymatic functions of
the cell.
Some of the selected organisms are grown anaérobically only with great
difficulty, and hence their chemical products cannot be readily investigated.
Other strains show the property of spontaneously agglutinating and cannot,
therefore, be very well shown to be derived from the original organism. In
this work, only those selected strains which, by means of the agglutination
test, could be demonstrated as related to the original organisms, have been
employed for the examination of the decomposition products from glucose
Glucose and Manmtol by B. coli communis. 475
and mannitol. Two varieties of selected organisms have been used—the
one (CI selected) produced from the typical B. coli (Escherich) No. CI, like
the organism of Harden and Penfold (1912), produced acid and no gas from
glucose, while it still produced gas from mannitol, and also retained the
power of decomposing formates in the presence of glucose (see Grey, 1914).
The other (CF selected), obtained from the organism CF, now gave acid but
no gas, either from glucose or from mannitol, and was also unable to decom-
pose formates even in the presence of glucose. This second selected organism
might perhaps be regarded as representing a further stage of selection than
the first, but I have not found it possible so far to obtain from CI a strain
comparable to CF (selected).
The Relationship between the Normal and the Artificially Selected Strains
as established by the Agglutination Reaction.
The artificially selected strains (when made into an emulsion by the
addition of normal saline to an agar growth) were found in many cases to
agglutinate spontaneously. The freshly selected strain was therefore first
plated out on plain agar and agar slopes made from several individual
colonies. By this means it was found possible to obtain strains which did
and strains which did not agglutinate spontaneously. The latter were then
treated with rabbit serum containing the specific agglutinins for the normal
strains CI and CF, and it was found that the serum obtained by inoculating
a rabbit with normal CI agglutinated normal CI and the artificially selected
Table I].—Demonstration of the Relation between the Normal and Artificial
Selected Strains (obtained by the Chloroacetate Method) by means of the
Agglutination Test.
Dilution of the serum.
Bacterial emul-
sion made with— | |
1/100. | 1/200. | 1/400. | 1/800. | 1/1600. | 1/3200. | 1/6400. | 1/12800. | 1/25600. | 1/51200.
Serum obtained with CF normal.
CF (normal) ...... ap ap ap |[ae sp ae lish qe ae ah ae ae) ap se ap | ae Se sp || cp ap or ++ ++ —
CFE (selected)...... ee[tealeeeleea| ees leee lata ++ ++ -
CI (normal) ...... - _ — — — — = = we =
CI (selected) ...... - = = = = 71 a | Th =r im
Serum obtained from CI normal.
CI (normal) ...... sake alee ay bastasis +++) 4 | = | |
CI (selected) ...... tet tr tittt] ++ + -
CF (normal) ..,.... = ae
CF (selected)...... = =
476 Mr. E. C. Grey. Decomposition of
strain derived from it, but did not agglutinate the strain CF. And, likewise,
the serum obtained by inoculating a rabbit with the normal strain OF
agglutinated the normal strain CF, and the selected strain derived from it
(CF selected) up to a dilution of 1 : 25600, but did not produce the slightest
agglutination with the normal or selected strain No. CI.* This may be seen
from Table IT.
Analytical Technique.
The methods of analysis described by Harden (1901) have been for the
most part closely followed; certain slight modifications, however, have been
introduced, which may be described here.
Volatile Acids—In the steam distillate which is used for the determina-
tion of formic and acetic acid, the formic acid has been determined by the
formation of mercurous chloride, and the acetic acid obtained by subtracting
the amount of formic acid so found from the total acids determined previously
by titration of the whole distillate with alkali, using phenolphthalein as
indicator. Two errors are introduced here due to the presence of small
amounts of carbonic acid and lactic acid in the distillate. The carbonic acid
has, therefore, been estimated by barium hydroxide and the lactic acid by
Ryffel’s method. This estimation of lactic acid in the distillate becomes of
importance when the amount of acetic acid is small.
The distillation to obtain the volatile acids was carried out in two stages.
The first fraction was obtained without admitting steam, measured about
400 c.., and contained the alcohol and part of the volatile acid. This
fraction was titrated with standard baryta solution. A slight excess of
baryta was then added, and the solution distilled with a fractionating column
in order to remove the alcohol. The residual fluid now contained a granular
precipitate of barium carbonate, which was removed by rapid filtration and
titrated at the boiling point with N/10 H.SO,. The barium hydroxide
corresponding to the barium carbonate was deducted from that required to
neutralise the first distillate. In this way an accurate correction may be
made for the carbon dioxide dissolved in the distillate.
After removal of the first 400 c.c. steam was admitted, and the distillation
continued until 100 c.c. of the distillate required only 0:-1-0:2 c.c. of normal
alkali for neutralisation. The total steam distillate usually measured about
2500 c.c. The distillate was neutralised with potash, united with the first
fraction, and the whole evaporated to dryness. The residue was dissolved
in 100 cc. of water, and an aliquot portion used for the determination of
* The agglutinating sera were kindly prepared for me by Dr. J. A, Ankwright of this
Institute, to whom my best thanks are due.
Glucose and Mannitol by B. coli communis. 477
formic acid, another portion being used for lactic acid by Ryffel’s method
(1909).
The extent of the correction for carbonic acid and lactic acid in the distil-
late of volatile acids may be seen from the figures
quoted in the table on p. 478.
Collection of the Gas.
The carbon dioxide and hydrogen evolved were
in some experiments collected in the apparatus
of Harden, Thompson, and Young (1910); in
other cases a simplified form of this apparatus
was employed, which is here figured. The object
of this modified form of apparatus is to dispense
with alltaps and to reduce the number of glass
joints. The present form of apparatus has but
one glass junction, and has also the advantage
that when evacuated it can be sealed by allowing
mercury to rise in the inner capillary tube through
which the air has been pumped out of the flask.
The arrangement for maintaining the pressure in
the fermentation flask constant (by adjusting the
level of the surface of the mercury in the reservoir
automatically) is also of a simpler type.
The flask A is evacuated by means of the
capillary tube, which passes up through the tube B
in which mercury rises as the air is removed.
The tube from the fermentation flask in the
incubator is attached to D by a rubber junction.
The gas in the fermentation flask is evolved
under atmospheric pressure, this equalisation of
the pressure in the flask with that of the atmo-
sphere being effected by means of the S-shaped
syphon (s), which is filled with mercury and
automatically adjusts the level of the surface of
the mercury in the mercury reservoir.
By plunging the rubber tube c beneath mercury
and opening the clip K mercury may be allowed
to rise in the capillary tube, and thus the flask A becomes completely sealed
from the atmosphere.
The neck of the flask A may be plunged beneath wax. This substance
478 Mr. E. C. Grey. Decomposition of
although quite effective, is somewhat troublesome to use, owing to shrinkage
on cooling. A rubber stopper plunged beneath mercury is, on the whole, a
simpler means of sealing off the flask.
Total volatile acid, Carbonic acid, Lactic acid.
¢.c. normal potash. c.c. normal baryta. ce.c. normal (Ryffel’s method).
48 °5 1°44, 1°68
38 2 1:0 itval
34°3 0°3 0'8
51°5 1°0 1°0
Al ‘1 1°3 0°6
Results of Analysis of the Decomposition Products of Glucose and Mannitol
formed by the Action of the Normal and Selected Strains of B. coli communis.
Table III.
Typical B. coli communis (rapidly motile) No. Cl.
Product. | Normal. | Selected.
| per cent. | per cent. Mean. |
On Glucose.
| 0] @ Paasentnans icongoasotna 14 90 14°74 14°82 | 2°25
I (iE epee. caer sepals nt 0°55 0°52 0°54 +18 66 0:08 $17 :23
UROBTMO — cosocovencoone 3°24 3°36 3°30 14 90
Acetic uistaiae cecerece 14:10 12°91 13 -00 5 69
Tiactre eee newssk csdone 39 *42 36 ‘91 38 :07 59 60
Succinicyereeeeseeaece 3 60 4 ‘60 4 20 5-50
All coholigeeeereee need 12°83 11 :02 11°93 4-90
| 85 *86 92 92
Ratio CO, : Hs 1°23 1°29 1°26 1°28
On Mannitol.
(OO yas aner ais aodnoreaanee: 26 -66 | 28 -00 2 BB} 118} Bs
1s [Re eT Gocabecnerns 1°04 1:06 1°05 $35°77 0°64 +31 °45
TOMA 555 snododnococe 7°21 7°56 7°39 | 17 °48
INCObIC! 24) deen cen sss 7°33 6°75 7-04 7:20
Tactic: cciseeees secant 22°82 26 °27 24°55 19 -95
Succinic ............++ 8 80 5 ‘00 6:90 8°61
INGO So soqasnsonnboe 27 06 26 *85 26 °95 27-46
101-21 | 94-67
Ratio CO, : Hy... 1:17 720 1:19 0-95
Glucose and Mannitol by B. coli communis. 479
Table 111—continued.
Variety B. coli communis (very slight motility) No. CF.
Glucose. Mannitol.
Normal. Selected. Normal. Selected.
per cent. per cent. per cent. per cent.
OW) casdauhauasneanpceene 16 ‘92 ] None 38 °50 None
Le Gye Rane OR PCA EERE 0-42 fae 07 None + 11°80 1°45 > 41°52 None > 32°50
TOWING Gaocedoosouesd 9°73 11-80 IL Sif 32 50
JANGEING, Ser eanpancnd aude. 18 -49 10-13 12°88 11°20
IGG YEnIO eee apeoroneoceogae 36 °83 62 -00 7-48 15 *84
SCONE erqodueesascede 0°74 0-80 5 ‘60 6-20
INCOMOH, hg sabaco cue eac 18 -06 5°30 26 °56 22°89
101 19 90 :03 94 04 88 °63
Ratio CO, : Hy... 1°83 1:21
These results may also be expressed as carbon atoms per molecule of
glucose and mannitol respectively.
Table IV.
|
| CI (rapidly motile). CF (slightly motile).
Product. | = i
| Normal. Selected. Normal. | Selected.
| per cent. per cent. per cent. per cent.
Glucose (carbon atoms per molecule).
COR eee, nisms 0-60 0°10 | 0-70 ba
IN@ITING --onbgoncaseAc0de0 0:14 0°55 0-40 0-46
ANCEWG croceashononseddes 0°81 0°34 1-11 0°61
1 DEXA ON padeber coceanrneee 2°37 3 60 2°20 3°72
SHECOWEKE cogcansoogoono0 0°22 0°27 0-04 0-04
ANIGOIIONL “cooeaboborob bce 0-90 0°39 1°41 0°40
5 “04 5°25 5 86 5°23
Mannitol (carbon atoms per molecule).
CORD rehsteesnansnsaces 1°09 i 0°54 i iL Aai3} I 3 ., —
Formic... 0-28 \ 1°37 0 aah 2s 0 cal nee 1-27
UNCOUIG ae sets eanitna isis 0°44, 0°45 0°78 0-67
BEN CN acc aeeer cerop acts 1°48 1°20 0°45 0°95
SGOT sgonodonedeouen 0°36 0°45 0°34 0°38
ANGAMOM! ceuabesagnovsse 2°11 2°15 2:07 1°86
|
5 “76 5°48 5 ‘28 5°13
480 Mr. E. C. Grey. Decomposition of
Discussion of Results.
The most significant fact in connection with these results is that whereas
in their action on glucose, the artificially selected strains of B. coli
communis have been considerably modified, in their action on mannitol the
only important change is the non-decomposition in the one case, and only
partial decomposition in the other case, of formic acid into carbon dioxide
and hydrogen,
The results with mannitol present greater uniformity than those with
glucose, and may be conveniently considered first. It will be seen that the
ratio (alcohol + acetic acid)/2: formic acid* is practically constant and almost
equal to unity. Thus
DE adie O08 MOG Ee 43285
253
cet ye reece Rp sie sy ep 6)
2x 137 7 SPaeaeos ae Secacoe "> Ox L27
This relationship also holds good for the earlier analyses of Harden (1901),
and points to two conclusions—
(1) Alcohol and acetic acid are probably derived from an intermediate
substance common to them both, and they may therefore, to a certain extent,
replace one another. (2) This intermediate substance from which alcohol
and acetic acid are produced is itself formed in constant ratio to formic acid.
Lactic acid might be regarded as being formed directly from mannitol by
the action of a special enzyme, but this could only oceur if (a) hydrogen
were evolved in excess of carbon dioxide, or (6) alcohol and formic acid
were produced by the same enzyme which produced lactic acid, as, for
example, in accordance with the equation
CsHi406 = C3H.03+ C2H;0H + HCOOH.
But if such a change as is represented by this equation were effected in one
step by a single enzyme then, since the proportion of lactic acid actually
produced is only one-third of that demanded by this equation (see Table IV),
it would follow that there must be another origin for alcohol and formic acid.
The foliowing hypothetical schemes are put forward to represent the
decomposition of mannitol and glucose :—
Mannitol, C,H,,O¢.
|
Y
Lactic acid, C3H,03. ——— Intermediate substance A + (2H)*
| nal
Formic acid, CO,:Hs. Intermediate substance B'****""*"* Alcohol, C,H,0O.
| :
1 ;
Alcohol, C,H,O. Acetic acid, C.H,Os.
* Hydrogen is here written as atomic hydrogen to indicate that it is intramolecular.
* Formic acid includes free carbon dioxide and hydrogen.
Glucose and Mannitol by B. coli communis. A481
Glucose, C,H,20,.
|
Y
Lactic acid, C3;H,O3. <5 Intermediate substance A.
Formic acid, CO,:H,. Intermediate substance B.
| ee
Alcohol, C,H,O. Acetic acid, O,H,0,,
The intermediate substance A is unknown, but is postulated to account for
the formation of lactic acid in such a way that the enzyme which produces
lactic acid from glucose may also produce lactic acid from mannitol. The
substance is probably related to pyruvic aldehyde.
The intermediate substance B from which it is suggested that alcohol and
acetic acid are derived is probably acetaldehyde. This view is supported
by the evidence that acetaldehyde may be detected among the products of
decomposition of glucose by B. coli communis (Grey, 1913).
Two molecules of acetaldehyde might undergo the Cannizzaro reaction
(Parnas, 1910) with the production of alcohol and acetic acid, thus
2CH;CHO+ H.0 = CH3;COOH + CH; CH2'OH.
If this were the main change in the case of glucose, it would account for
the production of alcohol and acetic acid in approximately equimolecular
proportions.
Again, acetaldehyde might be reduced to alcohol as postulated by
Kostytscheff (1912) for alcoholic fermentation by yeast or directly oxidised.
In the case of mannitol this reduction might be of great importance. Ii is
represented by the dotted lines in the scheme. And since, in this case
the whole, or nearly the whole, of the hydrogen formed, along with the
intermediate substance A, would be available for this purpose, the result
would be the production of alcohol in large excess over that of acetic
acid, which is actually observed.
While, however, the decomposition of mannitol and glucose may thus be
represented as occurring along the same general lines, it is clear that some
essential difference must exist between the mechanisms of the two reactions,
or they would not be so differently affected by the process of selection on
chloroacetate agar.
The simplest supposition is that this difference affects the production of
formic acid and intermediate substance B, for artificially selected organisms
produce these substances from glucose in greatly diminished amount, whereas
from mannitol their production is not seriously altered.
While the exact nature of the difference in the two mechanisms must still
482 Mr. E. C. Grey. Decomposition of
be a matter of conjecture, it may with some probability be supposed that
it is connected with the presence in the products from mannitol of hydrogen
available for reduction. It must be remembered that the two hydrogen
atoms possessed by mannitol in excess of those present in glucose are only
capable of reducing half the possible amount of B which could be produced
from one molecule of mannitol. Hence, even if- half the mannitol were
converted into lactic acid, these extra hydrogen atoms could be completely
taken up by B. As a matter of fact not more than one-quarter of the
mannitol appears as lactic acid, so that a considerable part of B is reduced
to alcohol and the remainder probably undergoes the same change as in
glucose, forming equimolecular proportions of alcohol and acetic acid.
It must be noted that from the above considerations one would expect
that the production of acetic acid from mannitol by the selected organism
would be somewhat less than by the normal. In my figures, however, this
is not demonstrated to be the case, but it must be remembered that the
amount of acetic acid produced by the selected organism does not exceed that
produced from glucose.
In the absence of more experimental results, however, it would be
premature to discuss other possible origins of acetic acid.
In the scheme for the decomposition of mannitol the production of the
excess of alcohol, as compared to the case of glucose, is represented as
occurring through the agency of this extra hydrogen.
In the case of glucose, on the other hand, alcohol can only be produced if
there be simultaneously the formation of some oxidation product, or in
other words the hydrogen would have to be supplied by a reductase.
It should be remembered that the aldehydomutase of Cannizzaro which
brings about the conversion in this case of two molecules of acetaldehyde
into acetic acid and ethyl alcohol is in reality also a reductase, the acceptor
for the oxygen being the same as the substance reduced. The essential
difference between the two changes would then reside in the necessity
for the co-operation of a reductase in the decomposition of glucose which
would not be required to the same extent for that of mannitol.
In all other respects after the preliminary decomposition of the original
molecule the two actions would then require exactly the same enzymes.
Considered dynamically, the reaction by which the intermediate substance A
changes into formic acid and substance B occurs more rapidly with mannitol
than with glucose, so that in the final products less lactic acid is formed in
the case of mannitol than in the case of glucose.
This acceleration of the reaction in the case of mannitol by which inter-
mediate substance A yields ultimately formic acid and alcohol as chief
Glucose and Mannitol by B. coli communis. 483
products, may be due to the reduction of substance B to alcohol whereby it
is removed from the sphere of the decomposition of A.
If, then, the reductase were to be diminished as the result of selection on
chloroacetate agar, the removal of B from the sphere of decomposition of A
would be slower. The decomposition of A into more of B and formic acid
would therefore be specifically hindered, and as a result the production of
lactic acid relatively increased.
This is what is actually observed. On the other hand the decomposition
of mannitol would be unaffected, as is also found to be the case.
The view that the artificially selected strain produced by growth on
chloroacetate agar is deficient in some reducing mechanism is further
supported by the fact that many of these strains show diminished power of
growing anaérobically. Moreover it might be expected that this method of
selection would lead to the survival of a strain deficient in reductase, for
a strain with a highly developed reducing mechanism would probably convert
monochloracetic acid to acetic acid with the liberation of hydrochloric acid,
which would certainly not be of advantage to the organism. Such a process
might therefore lead to the survival of the strain in which the reducing
mechanism was poorly developed.
Summary and Conclusions.
Two artificially selected strains of B. coli communis obtained by growth of
normal J. coli communis (Escherich) on agar containing sodium chloro-
acetate have been examined quantitatively as regards their action on glucose
and mannitol. In both cases the selected strains produced from glucose,
lactic acid in relatively greater, and alcohol, acetic and formic acid in
relatively less, proportion than did the original strains from which they
were derived, whereas from mannitol there was no diminution in the
production of alcohol, acetic, and formic acid.
From these results it is inferred that the artificially selected strains have
not lost the enzymes which bring about the final reaction in the production
of alcohol and acetic acid, but that the process of artificial selection has led
to an absence or diminution of the reducing mechanism of the cell so that
some intermediate substance, from which formic acid and the precursor of
alcohol and acetic acid are derived, cannot be readily decomposed.
In conclusion I wish to express my thanks to Prof. Harden, F.R.S., in
whose laboratory this work has been done.
484 Mr, kh. P. Gregory.
REFERENCES.
Grey, E. C., ‘ Biochem. Journ.,’ vol. 7, p. 359 (1918).
Grey, E. C., this vol., p. 461 (1914).
Harden, A., ‘Chem. Soc. Journ.,’ p. 610 (1901).
Harden, A., ‘ Journ. Hyg.,’ vol. 5, p. 488 (1905):
Harden and Penfold, ‘ Proc. Roy. Soc.,’ B, vol. 85, p. 415 (1912).
Karcezag and Méczév, ‘ Biochem. Zeitschr.,’ vol. 55, p. 79 (1918).
Kostytscheif, ‘ Zeitschr. physiol. Chem.,’ vol. 79, p. 143 (1912).
MacConkey, ‘Journ. Hyg.,’ vol. 5, p. 333 (1905).
Pakes and Jollyman, ‘Chem. Soc. Journ.,’ p. 386 (1901).
Parnas, ‘ Biochem. Zeitschr.,’ vol. 28, p. 274 (1910).
Penfold, W. J., ‘ Proc. Roy. Soc. Med.,’ p. 97 (1911).
Ryffel, ‘J. Physiol.,’ vol. 39, ‘ Proc.,’ p. v (1909).
On the Genetics of Tetraploid Plants i Primula sinensis.
By R. P. Grecory, M.A., Fellow of St. John’s College, Cambridge;
University Lecturer in Botany.
(Communicated by W. Bateson, F.R.S. Received March 3,—Read
April 30, 1914.)
The purpose of this paper is to describe certain peculiar results obtained in
the genetics of two “giant” races of Primula sinensis. Cytological investi-
gation has shown these giants, unlike the giant races already described,*
to be in the tetraploid condition, that is to say, that whereas in ordinary
Primulas the chromosomes are # (12) in the gametic and 2 (24) in the
somatic stage, in the tetraploid giants the chromosomes are 2 (24) in the
gametic and, as nearly as can be counted, 4x (48) in the somatic cells.
Nilsson-Ehlet and Eastt have shown that factors of similar property may
be reduplicated in the same zygote (or gamete), with various peculiar
numerical consequences not otherwise intelligible, notably the appearance
in certain F2-families of such ratios as 15D:1R, 63D:1R, and so on, when
in the ordinary case 3:1 would be expected. The occurrences to be
described in part recall this phenomenon; but, as will be seen, they are
* Gregory, ‘Camb. Phil. Soc. Proc.,’ vol. 15, p. 239 (1909) ; Keeble, ‘Journ. Genetics,’
vol. 2, p. 163 (1912).
_ +t “Kreuzungsuntersuchungen an Hafer und Weizen, I and II,” ‘Lunds Univ.
Arsskrift,’ 1909 and 1911; ‘Berichte d. Deutschen Botanischen Gesellschaft,’ vol. 29,
p. 65 (1911). - Q
¢ ‘American Naturalist,’ vol. 44, p. 65 (1910).
On the Genetics of Tetraploid Plants in P. sinensis. 485
accompanied by others at first sight entirely paradoxical (as, for example,
the fact that the ostensible recessive may throw the dominant), and the
whole series may be regarded as of special significance in view of the
association with the doubled condition of the cell-constituents. Moreover,
in the tetraploid Primulas, the reduplication affects not merely the factors
for isolated characters, but extends simultaneously to the factors for all
the characters so far investigated.
The tetraploid giants with which I have worked are of two distinct races.
One of these, which will be referred to as the GX race, consists of the
progeny of a plant kindly given to me by Messrs. Sutton and Sons. The
other race (GT race) arose in the course of my own experiments. Two
-non-giant diploid plants were crossed together reciprocally. The F, from
one of these crosses gave a perfectly normal F2, consisting of non-giant
plants among which all the expected classes of offspring were represented
in numbers closely approximating to expectation. The F, from the
reciprocal cross gave no seeds in a cross with one of its parent races and
gave only four plants as a result of self-fertilisation. These four plants
were giants, and from one of them the GT race has been bred.
Up to the present time, neither the GX nor the GT races of giants have
given any fertile seeds in crosses with various non-giant (diploid) races,
whichever way the:crosses were made. In this respect they differ from
a diploid giant race, with which I have worked, which proved quite
fertile with non-giants. It was this difference in behaviour which led to
the discovery of the tetraploid nature of the GX and GT races.
In the tetraploid plants the chromosomes are naturally more crowded on
- the spindles than they are in diploid plants, but in polar views of the
spindles of either of the maturation divisions there is no difficulty in
determining that the number of chromosomes is normally 24 (compared
with the 12 chromosomes found in diploid plants). In the somatic
mitoses the chromosomes are longer than those of the maturation divisions
and exact counts are difficult to make, but a number of counts have given
numbers approximating to 48. The maturation divisions sometimes show
some degree of irregularity, one or two chromosomes lagging behind the
others in the movement to the poles, but Iam not yet able to say whether
fertile germ cells having more, or fewer, chromosomes than 24 are ever
formed. There is no visible difference between the chromosome groups of
the thrum-eyed (short-styled) and pin-eyed (long-styled) plants.
The two plants which were the progenitors respectively of the GX and
GT races each possessed its own series of dominant characters, in respect
of which its origin would indicate that it was heterozygous. In the
486 Mr. R. P. Gregory.
course of breeding in the direct line from these plants the recessive types
have from time to time appeared. The course which this process of throw-
ing recessives has taken is shown in the following table :—
Dominant character of parent. Recessive character. Generation in which the
recessive first appeared.
: GX Race.
Petals cut at the edges Petals heart-shaped with sim- FB
(sinensis type) | ple median notch (stellata
variety)
Dominant white..................| Magenta flowers.............-..0. F,
Green stigma ...............0000 |) sRvecUB Ei pmmalem ences c-creeeaee F;
Mialgentian ty ccsasctunsrncectoe teens | Ried oss canest ouslentios tveceeene aes F,
Palmate leaves .................. Hernijleaives:. .euetassetd-. neon eee FE,
GT Race.
Dominant white ............... Macentaiiis iste eee eee F,
WHEY ES conons sganbonen ssa ndudebee: Riedie vcessnsncusce tosccnsarers FB;
Thrum-eyed (short-styled) ...| Pin-eyed (long-styled) ......... FB,
IRedystemisy 5s eevee. <0. ees Greenystemsyecepeee tetera F,
In the character of the petals and in “dominant white” the dominance
of the positive factor is not quite complete and the heterozygous plant
can be distinguished from the pure dominant by inspection. In each of
these cases, the appearance of the pure recessive is given in the table
above, and in each case the heterozygote was recognised in the preceding
generation.
In the GT race one expected recessive type, the double flower, has not yet
appeared. But in F3 two plants with semi-double flowers were obtained,
both of which would no doubt have produced doubles among their offspring,
had they not unfortunately succumbed to the attacks of fungus before they
ripened seed.
It is obvious that some of the foregoing recessive characters have made
only a belated appearance in the progeny of the original heterozygous
plants. Both races of the tetraploid giants, however, produce a relatively
very small quantity of seed in self-fertilisation, so that the families raised in
each generation have nearly always been small. Consequently, in the
present state of our knowledge of the processes of segregation in tetraploid
plants, one cannot regard the sporadic appearances of the recessive types as
providing a clear indication that processes other than the normal ones are
involved.
Besides the recessive types, both races of giants have thrown some
peculiar intermediate forms, which are distinct from any intermediate or
other forms known to me in the non-giant- diploid races. The characters, in
On the Genetics of Tetraploid Plants in P. sinensis. 487
respect of which giant intermediates have been produced, include both
morphological characters and colour-characters. They are—
Dominant Character. Recessive Character.
(1) Petals cut at the edges. Petals heart-shaped with simple
median notch.
(2) Tie-ring habit of the inflorescence. Inflorescence condensed.
(3) Palmate leaves. Fern leaves.
(4) Dominant white. Coloured flowers.
During the present year there have also been obtained some flower-colours
which are intermediate between magenta and red, but, as the diploid races
also produce certain colours which it is difficult to classify, further experi-
ment is necessary to show whether or not the new kinds of colour are
peculiar to the tetraploid races.
With regard to the characters (1), (2), and (3) above, it should be pointed
out that dominance is incomplete in the diploid races, but the giant
intermediates form a distinct class from the common heterozygous type,
which also occurs in the giant families, alongside of the peculiar intermediate
types.
The intermediates between the palmate and fern leaves are, however, the
most striking, because in the diploid races the dominance of the palmate
shape is, for practical purposes, complete.
In all the cases there is some range of variation among the intermediate
forms, and there may be differences of degree between the different organs of
the same plant.
Further, it has been found that in the tetraploid giants certain types of
flower-coloration may occur, which closely resemble the colours of certain
diploid pure races, but are, nevertheless, the product of a different set of
factors. This may be simply illustrated in the case of a Giant Red with red
stigma, which almost exactly matched the colour of my Red Stellata non-
giant race. The non-giant race is quite pure, and contains no colour-
inhibiting factors. The giant red, selfed, has given (1) forms like itself,
(2) more deeply coloured forms, and (3) pure and heterozygous “Duchess ”
types, that is to say, types showing the possession of the factor which
inhibits the production of colour in the peripheral regions of the flower.
Other similar cases have occurred, both in plants with green stigmas
(i.e. possessing the factor which inhibits colour in the central parts of the
flower), as well ds in those with red stigmas. These cases, then, provide the
striking result that the coloured form is shown to be capable of throwing
the dominant white.
VOL. LXXXVII.—B. 20
488 Mr. R. P. Gregory.
Phenomena of the kind just described, taken together with the fact that
the tetraploid giants have produced intermediates peculiar to themselves,
suggest some considerations as to the factorial constitution of the tetraploid
plants. Both classes of phenomena can, I think, be explained by means of
the hypothesis that, as compared with diploid plants, the tetraploid plants
possess a double set of factors. Since in the zygote of a diploid pure race
each factor is to be regarded as represented twice, AA, it follows that the
tetraploid plant, according to this hypothesis, will be AAAA, and the
gametes from which such a zygote is formed must be AA, that is to say,
the factor will be represented twice in the gamete, instead of once, as it is in
the gametes of the ordinary diploid race.
Heterozygous tetraploid plants may, then, be any one of three possible
kinds, AA Aa, sg Aaaa, Since each gamete will contain two of the four
units (“ presences ” or “ absences”’) which make up the tetraploid group, the
gametes produced éy the three kinds of heterozygote, and the resulting
progeny in Fs, will be as follows :—
Case [—Heterozygote, AAAa; gametes, AA, Aa;
Fo, 1 AAAA : 2 AAAa:1 AAaa.
No pure recessives in Fo, but, of every four plants, one will give pure
recessives in F; in the proportion of one recessive in ie 16 plants (see
Case 2).
Case IJ.—Heterozygote, AAaa; gametes, AA, Aa, Aa, aa;
Fo, LAAAA :4 AAAa: 6 AAaa: 4 Aaaa : 1 aaaa.
F; contains one pure recessive in every 16 plants.
Case IJI.—Heterozygote, Aaaa; gametes, Aa, aa;
) fo) ? ey is) ?
F., 1 AAaa ; 2 Aaaa: 1 aaaa.
F, contains one pure recessive in every three plants; no pure dominants,
but one plant in every four will give pure dominants in F3.
Of the various kinds of heterozygote shown in the foregoing scheme, one,
namely AAaa, has the same proportion of positive and negative elements
(“ presences” and “absences”) as the ordinary diploid heterozygote. With
regard to the characters in respect of which the tetraploid giants have
produced peculiar intermediates, it is suggested that the intermediates may be
either AAAa or Aaaa. The former would presumably show the cumulative
effect of the three factors, like that which Nilsson—Ehle and East have
recognised in some of their cases, by giving a type more closely resembling
the pure dominant than does the ordinary diploid heterozygote, but in the
Primulas such types have not yet been definitely recognised by inspection.
489
. SINEUSIS.
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490 Mr. R. P. Gregory.
The intermediate Aaaa would be expected to show the dominant character in
less degree than the normal heterozygote; it is to this class that the
intermediates already described are to be assigned.
The intermediate characters do not, however, provide the most favourable
opportunity for putting the hypothesis with which we are dealing to a critical
statistical test, because the range of variation among the intermediates is
sufficient in some families to make classification by inspection a matter
of difficulty. This difficulty will no doubt decrease as one becomes more
familiar with the new forms, but for the present all that can be said is that
the results of the experiments are in general accord with the present
hypothesis.
A more critical test is, however, provided by some experiments relating to
the characters of thrum-eye and pin-eye, and red stigma and green stigma. In
these cases no intermediates have as yet occurred and it may be assumed
that one “dose” of the factor is sufficient to bring about the development of
the dominant character. The results of these experiments are set out in
Tables I and II.
Table I1—Crosses of Green Stigma and Red Stigma.
| |
F, x self. F, x recessive.
F, plant. = ==
| Green. | Red. Form of cross, Green, Red.
|
| 793/13 13 0 © |_- 725/18 as 2 | 5 2
72/13 | 17 @) | 72°/13 as 2 28 6
728/13 | 1 0 725/13 as 2 | 14. 4
681/13 44 2 | ggii3aa9 | . 15 1
| Ditto i 0)
Ditto | 3 10)
| 68/13 as g 48 17
| | |
In these results two kinds of heterozygous F, plants are clearly shown to
exist. Thus, in the crosses of thrum x pin (see Table I), the F, 681/13 gave
41 thrum 1 pin when selfed, and 65 thrum 15 pin when crossed with the
recessive. These numbers may, I think be regarded as representing
respectively the ratios 15:1 and 3:1, and the F,-plant may therefore be
identified as AAaa. The thrum parents from which the other Fi’s were
obtained each gave a small number of recessives in the Fj-families from
crosses with recessive plants. It is, therefore, not surprising to find that.
most of the F, thrum plants derived from their crosses have given F»’s
approximating to the lower ratios 3:1 and 1:1; that is to say, the majority
of the Fy’s are of the constitution Aaaa.
On the Genetics of Tetraploid Plants in P. smensis. 491
To turn to the crosses of green x red stigma (Table II): it should first be
pointed out that the GT race sprang from diploid races pure for green stigma
and no red stigma has ever appeared in this race bred in the direct line.
Plants of this race may, therefore, be written GGGG. It is entirely in
accordance with this that the F,’s from crosses of this race with red stigma
have all proved to be of the type GGgg, giving the ratios 15: 1 when selfed,
and 3:1 when crossed with the recessive. Heterozygotes of the type Ggge
have, however, been found by selfing green-stigma plants chosen from
families in which some of the plants had red stigmas. Ten such plants
have given altogether 99 green stigma, 34 red stigma.
It will be noticed that the Fy’s which appear in the green x red-stigma
crosses also appear in the thrum x pin crosses. The F) 681/13 is giving the
same ratios in respect of each pair of characters, namely, 15:1 when selfed,
and 3:1 when crossed by the recessive. But the Fy’s 72/13 are giving 15:1
and 38:1 for green and red stigma, and 3:1 and 1:1 for thrum and pin.
Taking the two pairs of characters together, and assuming for the moment
that there are no special inter-relations between the factors, these would
give the curious ratios of 45TG:15tG:3Tg:1tg when the F, is selfed,
and 3:3:1:1 when the F, is crossed by the double recessive(tg). The
actual numbers obtained are 28TG:4tG:0Tg:0tg in the former case,
and 22 TG: 25 tG:2 Tg: 10 tg in the latter.
In the foregoing results the different kinds of heterozygote stand out
clearly identified by their progeny, and, although there are considerable
discrepancies in individual cases, yet the general trend of these results
clearly shows, I think, that the tetraploid plants are endowed with a double
set of factors, as compared with the diploid races.
As has been remarked, the results of experiments with the intermediate
types are in general accord with the idea that my existing intermediates are
heterozygotes of the type Aaaa, in which one “dose” of the factor is not
sufficient for the full development of the “dominant” character. The
variations among the intermediates themselves, which are probably of the
same nature as the variations exhibited among heterozygotes in cases where
dominance is imperfect, are, of course, still to be explained. It is curious to
notice that when there is any marked variation between the organs of the
same plant it appears generally to take the form of a gradual retrogression
towards the recessive character in the successively younger and younger
organs, the effect of the positive factor being a little less pronounced in each
new organ formed.
In conclusion, it must be remarked that the results so far obtained do
not of themselves throw any direct light on the problem of the possible
VOL. LXXXVII.—B, 2?
492 On the Genetics of Tetraplord Plants mm P. sinensis.
‘
relationships between the factors and the chromosomes. Although the fact
that the duplication of the chromosomes has been accompanied by a dupli-
cation of the series of factors may seem at first sight to suggest a definite
connection between chromosomes and factors, yet, on the other hand, the
tetraploid number of chromosomes may be nothing more than an index of
the quadruple nature of the cell as a whole. The case is, in fact, exactly
analogous to the ordinary zygotic cell, which has 2% chromosomes and in
which each factor is represented twice. But there are grounds for
believing that further experiment with tetraploid plants may have a
direct bearing in this connection, for some of the experiments have
already given an unmistakable indication of the existence of special
inter-relations (in the form of coupling or repulsion) between certain
factors in the tetraploid Primulas. The work has not yet gone far enough
to permit of any useful statement of the results, but it is obvious that it will
provide a new opportunity for the study of the mutual relations between
factors in heterozygous plants, particularly as to whether or not special
inter-relations may occur between the two factors of the same kind (ue.
between A and A’), and as to whether either of the factors of one kind may
have relations with either factor of another kind (ze. A with either B or B’,
and conversely), or whether the A and B factors form one pair of related
factors, the A’ and B’ an independent pair, so that A may have special
relations with B but none with B’, and conversely.
Part of the expenses of this work have been defrayed by grants from the
Royal Society and from the British Association. I wish also to express my
great indebtedness to the authorities of the John Innes Horticultural
Institution for the facilities for work and the help they have so freely
given me.
493
Description of a Strain of Trypanosoma brucei from Zululand.
Part I.—Morphology.
By Surgeon-General Sir Davip Bruce, C.B., F.R.S., A.M.S.; Major A. E.
Hamerton, D.S.O., and Captain D. P. Warson,* R.A.M.C.; and Lady
Brucsg, R.R.C. (Scientific Commission of the Royal Society, Nyasaland,
1912-14.)
(Received February 24,—Read March 26, 1914.)
[PLatEs 21-23. ]
INTRODUCTION.
In July, 1912, Dr. A. Theiler, C.M.G., Director of Veterinary Research
(Union of South Africa), Pretoria, sent this Commission several blood
preparations taken from horses and dogs supposed to be suffering from
nagana. Much to the surprise of the Commission, a large percentage of
these trypanosomes showed posterior-nuclear forms. This disposed of the
contention that the so-called Zrypanosoma rhodesiense could be distinguished
from other species of trypanosomes by this peculiarity, and first led the
Commission to suspect that 7’. rhodesiense was in reality 7. brucev.
Dr. Theiler was then asked to send the living strain through to the
Commission in Nyasaland, and this, after several failures, was at last
successful.
The history of the strain is as follows: Mr. A. W. Shilston, Veterinary
Research Division, Pietermaritzburg, writes that it originated in a mule
which was naturally infected at Somkele in Zululand. A dog was inoculated
from this mule and brought to the Veterinary Research Laboratory at
Pietermaritzburg, where sub-inoculations into a series of animals were made.
Mr. Shilston says there is no possibility of this strain having been mixed
with any other, as, at the time he was working at it, it was the only species
of trypanosome maintained at the laboratory. He also states that he—in
order to prove that he was dealing with a single species of trypanosome and
not with a mixed infection—infected rabbits with single parasites, and the
resulting infections showed the same morphological characters as the original
strain.
From Pietermaritzburg the strain was transferred to Pretoria. Mr. William
Robertson, acting director during the absence on leave of Dr. Theiler, informs
* Major Harvey, R.A.M.C., resigned from the Commission and left Kasu, September
16, 1913. He was succeeded by Captain Watson, R.A.M.C., who arrived November 2,
1913.
VO UXRXCV Bs 2Q
494 Sir D. Bruce and others. Description of a
the Commission that the strain was kept going in Pretoria in horses and
cattle, in which animals it produced the typical clinical symptoms and
post-mortem lesions associated with nagana, and that it was always regarded
as a pure uncomplicated strain of 7. brucet. The thanks of the Commission
are due to Mr. Robertson for his perseverance in sending inoculated animals
to their camp at Kasu. Like the Japanese general outside Port Arthur, as
one batch succumbed he sent on another, until at last he succeeded.
The exact length of time this trypanosome was kept going at Pretoria
before being sent to Kasu is not given, but the information has been asked
for, and will be placed on record as soon as obtained.
In the opinion of the Commission, the trypanosome dealt with in this
paper is the same as that discovered by Bruce in Zululand in 1894, and
named 7. brucei by Plimmer and Bradford. Somkele is in the same district
in Zululand as that in which this species of trypanosome was first discovered.
In this paper the old Zululand strain will be called the 1896 strain, that
being the year in which it was first described; the new strain, the 1913
strain, the year in which it was received from Pretoria.
The Zululand trypanosomes were described by Bruce in his original
paper* as hematozoa which vary among themselves a good deal in size and
shape. Photographs were also given, which show a distinet dimorphic type.
In a later papert Bruce gives measurements of 200 trypanosomes taken
from preparations which had been made in Zululand in 1896, and also gives
six figures taken from the same source. From these it will be seen that
the trypanosome dealt with in Zululand in 1896 was a markedly dimorphic
form, with long and slender, intermediate, and short and stumpy forms.
From the above measurements and figures there cannot be the slightest
shadow of doubt about this.
In 1896 Bruce sent this trypanosome to England, and it was at once
placed in the hands of Kanthack, Durham, and Blandford by the Royal
Society to be reported on. Their investigation lasted two years, and was
published in vol. 64 of the ‘ Proceedings’ of the Royal Society. In regard
to the shape of this trypanosome they state that “the Nagana parasites vary
considerably both in size and form ; they may be long and pointed or blunt-
ended and somewhat stouter; some individuals are short and thick, with a
short flagellum, their protoplasm being crowded with rounded granules.”
No one who reads Bruce’s ‘ Progress Report’ and compares it with Kanthack,
Durham, and Blandford’s 1898 report can doubt that the same trypanosome
was being dealt with. This trypanosome was distinctly dimorphic.
* ‘Further Report on the Tsetse-fly Disease, or Nagana, in Zululand,’ 1896.
t+ ‘Roy. Soc. Proc.,’ B, vol. 83, p. 9 (1910).
Strain of T. brucei from Zululand. 495
About this time (1898) the trypanosome was handed over to Bradford at
the Brown Institute and named 7. brucei in a paper written in 1899.*
That the trypanosome named by Plimmer and Bradford was the same as
that sent to Kanthack, Durham, and Biandford in 1896 there can be no
reasonable doubt, There was no other species of pathogenic trypanosome
in any English laboratory at the time, with the exception, perhaps, of
T. lewisi, with which there could be no confusion.
Now, having shown that the original Zululand strain was a well-marked
dimorphic type of trypanosome, let us see how it compares with the 1913
strain.
Mr. Shilston kindly sent the Commission a description of this strain made
immediately after it had come from Zululand. He states that in the living
condition the variation in size and shape of the organism can be observed,
the long, slender flagellated forms being readily distinguished from the short,
stumpy forms, while all gradations between these two can be found ; that
the circular vacuole close to the micronucleus is very distinct ; and that,
although the organisms are actively motile, their progression is not rapid
and frequently they simply travel in a small circle.
Cuart 1.—Curve representing the Distribution, by Percentages, in respect to Length, of
400 Individuals of 7. brucei, Zululand Strain, 1913 (Shilston’s measurements).
(ya LT TET)
L| [13 [14] 15] 16] 17] 18] 19] 20] 212228 [24 [25 [26] 27| 26] 29]30 | 31]52]53]54]55)
et LL | |r a Eee
|e ae a NE | [ofa | a
el | ele ai a ene ea hn a Ja
fae ot tte ER
* “A Preliminary Note on the Morphology and Distribution of the Organism found
in the Tsetse-fly Disease,” by H. G. Plimmer and J. Rose Bradford, ‘Roy. Soc. Proc.,’
vol. 65, p. 274,
496 Sir D. Bruce and others. Description of a
Mr. Shilston also made a large number of measurements of this strain.
One of his charts gives the percentages in respect to length of 400 trypano-
somes occurring in the mule, dog, and guinea-pig, the measurements
being made at varying periods of the disease. This chart is reproduced on
p. 495.
In a previous paper* a curve will be found representing the percentages
in respect to length of 200 individuals of the original strain of 7. brucei,
measured from old Zululand preparations which had been made in 1896 and
were still extant. The numbers making up these two curves are doubtless
small, but they are fairly comparable.
Cuart 2.—Curve representing the Distribution, by Percentages, in respect to Length, of
200 Individuals of 7. bruce?, Zululand Strain, 1896.
ERG M al.) Ee Oo ge
& {13[14]15]16|17/18119 ]20[ 21] 22] 23]24[25]26 | 27]28/29|30| 31] 32] 33|34]35|
BEECH EEE EEE EEE
OE eee
BRS (RRR eee
DecoCceeecec ceo coesso
RRR ARR RRR eee
DERE ROR
’ Percent
Now although too much weight must not be placed on this comparison,
still it must be confessed that the two curves are remarkably alike, and
afford a strong argument that Shilston recovered from the Somkele district
of Zululand the same species of trypanosome which had been discovered
there in 1894. Again, when the action of these two strains on various
animals is compared, the same likeness is seen.
Bruce’s 1896 strain killed two horses in 30 and 49 days.t Shilston’s
* “Roy. Soc. Proc.,’ B, vol. 83 (1910).
+ ‘Further Report on the Tsetse-fly Disease, or Nagana, in Zululand, 1896.
Strain of T. brucei from Zululand. 497
1913 strain killed one horse in 35 days. The former strain killed five dogs
in an average of 21 days, the latter four dogs in an average of 19 days.
Taking these various arguments into consideration, it may be assumed that
the strain of trypanosome which forms the subject of this paper belongs to
the species 7’. brucei, a well-marked dimorphic type of trypanosome.
The object of this paper is to describe as fully as possible the morphology
of this new strain of 7. brucec from Zululand, in order to try to prove its
identity with the trypanosome causing disease in man in Northern and
Southern Rhodesia, Nyasaland, and German and Portuguese East Africa.
The importance of this cannot be overrated. It has been the habit in the
past to consider 7. brucei harmless to man, but if the above conjecture proves
to be true, then all Glossina morsitans areas where wild game and T. brucei
co-exist must be looked upon as dangerous. Evidence is accumulating
that this is so. Recently two Europeans have fallen victims to the tsetse-
fly disease in the Sebungwe district in Southern Rhodesia, a remote, savage,
unfrequented spot swarming with game and Gf. morsitans. This year also—
1913—as had been anticipated, several cases have been found in the Nyasaland
fly-areas to the north and south of the “ Proclaimed Area,” one case occurring
in a native village within a few miles of Zomba, the official capital.
In future papers, the Susceptibility of Animals to this Strain, its Develop-
ment in G. morsitans, Sera Reactions and Cross Inoculation Experiments will
be dealt with.
IJ. MorpHonocy oF T. BRUCEI, ZULULAND STRAIN, 1913.
A. Living, Unstained.
In the living and unstained preparations the dimorphic characteristics of
this species can be readily made out. The parasites are actively motile but
with restricted translatory movement.
B. Fixed and Stained.
The blood films were fixed, stamed and measured as previously described
in the ‘ Proceedings.’*
Length.—The following table gives the length of this trypanosome as found
in the monkey, dog, guinea-pig and rat, 1000 trypanosomes in all.
* B, vol. 81 pp. 16 and 17 (1909).
498 Sir D. Bruce and others. Description of a
Table I—Measurements of the Leneth of 7. brucei, Zululand, 1913.
|
|
ag | | | In microns.
0.
Date. Of jl a)sBalinal gal gee eee all ae eee
| expt. | aac | staining: | Average | Maximum) Minimum
| | length. | length. length.
|
io) 1918;
Hebel O Were: 1833 | Monkey ...... Osmic acid | Giemsa 21 °4 27-0 16-0
Wieieks WON 1835 hats Ah 9 y | s | 23-2 29-0 18 ‘0
a Oe! 1836 Caoae oe se \ apd se | 23-4 30-0 18-0
ees Ore 1857 Nh, ay - ts | 21-9 31-0 170
Wee pil Sines 1834 ifn muilt.s i$ f | 20°3 28 -0 160
Pee ase el| 1835 emai ta) % ts | 19-0 24-0 12-0
[iene aL SIS Se | 1836 ede ENE 4 f i 20°1 28-0 16:0
so Meets cles 1834 popes Renee 5 is 20 6 27°0 170
Ao Ohe Me 1904ul Dopmee amen « i 22°8 29 -0 19-0
Reso Once 1905: | ett oe. is ‘ 20°5 29-0 17:0
Rea Osan re 1907 ap AREER STs bs 55 | 20°6 280 |) GE
me20N TOUS Saet, che ort as re a uy 30-0 180
er Deke NOGA ee ce were ‘< % 251 31-0 190
ad Dae 1905 || A sf fs 211 25-0 18-0
Oy NaN A LSO7e ee eee - . 20-0 30:0 | 160
ae OY T9084 ym tee ee <3 é 21-2 25-0 18-0
er ee 1904) | er teuh teet o 53 i. 21-7 30-0 18-0
Ben DT NGOS & Me Pee " # | 21°8 31-0 18-0
See TOOG)| AS a i | 20°8 28-0 18-0
ae TOOTE IE erase | af . 19°1 21-0 160
py aU sence 1908 Pewee oem meee | ef "6 24.8 32-0 18:0
date te 1844 | Guinea-pig ...| i FE | 27 °6 350 18-0
AO eee te 1894. 3 a is “ | 21-0 29:0 |) 1 isso
i ae 1843 “ . 34 - 21-9 29:0 | 17-0
Be Ae ra 1894. e x x | Bile 31-0 17-0
1 aS ee 1829) Rate... a is 22-9 28 -0 17-0
i, Se Tey eae ae Se On is i 22-5 28-0 18 0
We SPORE, eden oe ‘s a) 21-5 26-0 18-0
5 Oe 1G 20)1|eeo hes. Aken i ‘ 21-6 25:0 | 17°0
cv ROL ae TFS) ta ace a NE | i i 20°9 25-0 180
mee TO20N | eee oe ‘i os 20°7 24-0 17-0
ay) LO. 1829 Pree caccano ues - . 21:2 24-0 19-0
Oras 1829 SM GARTEN y 3 21 °6 23509) |) SeLSEO
S10 eae 1S291 |e ceeds | x x 21°3 24:0 | 17°0
Page| il bea 1829 pa Shei omesteaicy = op 19 °8 25°07 | ie)
Rae Wy rs 1829 Be aerate a 55 20°5 22-0 18-0
= ee 1S29%| bse Reo ,, “a 2029 -| = 25:0" |) A8ep
59 Pe 1829 gr ictonatatenal os 0 20 °6 23505) ||P ee lSeo)
er ae ae 1829°| a meee . % 21-7 28-0 18-0
ee, SON ts 1SD9) |h ee ser eat Sere . . 21°3 25-0 19-0
ae (Sis sears 1829 Prpcedonacaccte si 90 20°5 23:0 | 18:0
paint itis: 1829 pS Se een As 20-0 24-0" 7-0
Be US bs on B29 4g ate ees ae % 2 20-2 23:0 | 17:0
Spe hAa ce 1829 55. MesaMani seis op 1p 20°6 26-0 18°0
PAT WANs. es eT 2O | Weare aah tee! i ve 19°5 23:0 a geG
RE I te TS29),) pie ce ameeener . i | 20-0 22:0 | 17-0
Stl ance ' 1829 oe ee eee a! i< 20-4 23 0 18 0
ge KT cee GOON) esnemu ee mee ie 5 19°7 240 17-0
hp ast ae S20, tees eee ae | 5 i 20-2 2-0 18 ‘0
5 LORE 1829 Srimnoraaaescnc0 | 0 op 19-6 22-0 18-0
| |
21°0 35 °0 12°0
Strain of T. brucei from Zululand. 499
The average leneth of 7. brucei, Zululand strain, 1913, in the monkey, dog,
guinea-pig, and rat, taken from Table I, is as follows :—
Table I1.—Average Length of 7. brucet, Zululand Strain, 1913.
In microns. |
Number of 4
Species of animal. trypanosomes |
measured. Average Maximum Minimum
length. length. length.
Monkey) se.-es-<-2-- 160 211-2, | 31-0 12-0
DOYS ceaoodsoosupedetne 260 21°5 32 °0 16-0 |
Guinea-pig ......... 30 22 °9 35 0 170
ISRIB. oacaoadenris acnade 500 20°8 28 °0 17°0
The above table shows that a good deal of difference in growth takes place
in different animals. Compare, for example, the guinea-pig with the rat: the
former with a maximum of 35 microns, the latter with a maximum of only
28 microns,
The next table gives in detail the distribution in respect to length of
1000 trypanosomes. The Commission feel hardly justified in taking up space
for this purpose, but it is thought that perhaps in some unknown way these
figures may be of use to the statistician.
Cuarr 3.—Curve representing the Distribution, by Percentages, in respect to Length, of
1000 Individuals of 7. bruce?, Zululand Strain, 1913.
nema Nlierons sh |
Es) [12| 13] 14] 15] 16] 17] 18]19|20 [21 ]22[23 24 [25]26[27] 28 [29 [30]31 [3233 ]54]55|
Percentages ~
ule ar calles
et
I aa
a
pao sav
et [a [a a Sse
ge |e a
This curve is made up of measurements from 160 specimens of trypanosomes
taken from the monkey, 260 from the dog, 80 from the guinea-pig, and 500
500 Sir D. Bruce and others. Description of a
a :
ao WANIOAMWOAOGODWOMAAOAMDDHADOSDHDNS 10190 OE
SS ey ANMHOROONOCOMHORTHOOHRHARHANHHOS
as ANANANAAANAANNAAANAANAMANAANANANQANAIA
: 8 ee near eee elie ae eel eee memlbe si I lel tI
2 alainy
& 2 alee aa RT WA Mtomlemietiai te) he) |
ss & Fae eal ET Ta Tee eae eae See
nl
3 eee ee.
cS :
3 $3 alee tel Peas leat! epee ok
rant o 7 a aad ; a. ae ae > aie 7. .
5 5 Wel rant ee Pa pS ea
& ee re ee IE
Lm} .
= % | La |e S| ae
XQ ai as
-s R SPT A ae Pee a) ies? Ses ssa a
> oG [Sa pain | Srinin [| | aso [Ss Se
Ss nN
SI eo i iar Mice babieicts ae] i i |
a4 ——— eee
(o) .
e R | (oes ise Ae
os} 6 ae
2 gf) sees en lt ar
“4
ue ‘ oo] NAH aia aH Nj) | ja alten Sica! al)!
rg a | a | ¥sliarall [falas lah Pat (eae
=! z $8 | FA] Aa] | | OANMANAwO |] | | | |] | HAT |
—
|
is NSN | AADMTOMHHRAHAD | RAMANA 4 AH Ho OH
7) nN | | | |
=
= = AP APPA AO MAON TION ria | | Wi | cucyco wm cc
io]
)
al S AN AO MOON AAA A HCH OD OOD IO | IOAN ed 419 a9
Ss
a) a ANANAA WOW AG | MOANA AOOOMAONIDIAH ADM
Q,
n i}
se cs | FANT | [wT PATA N OTA oNOt | aN |
i=} _
a =
s 2 [Psfee rete eS SS TMM STS lS ae ae
aS) 25 x = yee 8 ME aed
= Ss Th [sk leah ees Pet eonc le ea eas cme hails]
ro J
5 Ppa he ha berlin feel Po eee a SO MMPPL NAb iabes ij |
RB |
iT an —— = —
7 See aS lat oll TTT ST oD PSSST oS aT eal ant
At = ele eehey
= of hashes elie 5 sae Ol et tea al SAE" Vatam ap Pl
at ke
= s Fabel el [Dalek Skala teste te ea at
s : Bh. 2.5 a
Ee : bp
i 7B :
q s site al a:
A GE RRFRRAR Rey “a z
Sr ai en ed! SRE) Yfke ee eae i Fait eat ence ed tf ed a PY Mae Ch
a S SISO ah Sa aA SS oes GBS Sia 5
Strain of T. brucei from Zululand.
water geen hier ie a er Mae ker ea imple
FH BODSCHHOSOODOaDOROGD
NNNANANANANANANANAAR AS
0°2
4 =~
| Ts (he TF elidel fl foal all lel eM ae S
eo
peal bobebel I fepilicshadliork. ce ebes Tiel A es
nN
meek tts Frode Pet Cel eal nt ba ee ss
4 =
tell Ted | ie haladiehst a
ee eae eee el = Ss
| ror) °
mieiel | el | | pea plea S
~
ale belle eal elie PR = ‘
a = ¥
tet el eb de led lhe delet a ed
=) 2
Bo Mla tet ee oe R a
roe) 2
eee eer a ir ES B
a)
Paleiies poo hae ele de Li bhied). Eg A x
~
01 holies] Ota fl 8 le i nT eS) Ba
APA] | NOONAN | AG | Sa.
leas’ §
HOA | WHA FANRIIAH | Ho er Be
art H(i
Tn)
COPMNWWMAFOWNWNMOFAD0 Ww = =
o a =
DHANWOONATIANIOMMNDOIO eS iz
Loa re
EA
A | OMMFANNATAANM OIDs 8 iS
Lan 4
°
| [ANF OH | MONAHAN AW s o
Len =
ST Ep ae ee ae | | ee
fel ig iol hela El Sa eg) sles
Cape Wea Lon
oat heal | CP Wh = =
be] a
PER TR Mot on Mow inca etl oq) lon, fod et ox ren! jin cm
RA RRRRRARR AR FARR RRA
ETOH) essences
Percentages NOM |
501
502 Sir D. Bruce and others. Description of a
from the rat. It is very similar to some of the curves taken from the
Nyasaland human strain:* compare Strains IT and V. But, on the other
hand, it is very unlike some of the others, as for example Strains I and II.
Cuarrt 4.—Curve representing the Distribution, by Percentages, in respect to Length, of
500 Individuals of 7. bruce’, Zululand Strain, 1913, taken on nine consecutive days
from Rat 1829.
eM MGR ese bel ee
Me ptotonoomencroore
SRE ERA
BOGRRecec cee esas
tt tt Fy ft
| |e] | |
2
SPS Se ete
2 Denese mms
This is rather a peculiar curve, but is not unlike curves obtained in a
similar way from the trypanosome causing disease in man in Nyasaland, as
the following chart will show :—
* ‘Roy. Soc. Proc.,’ B, vol. 86, pp. 285-802.
Strain of T. brucei from Zululand. 503
Cuarr 5.—Curve representing the Distribution, by Percentages, in respect to Length, of
500 Individuals of the Trypanosome causing Disease in Man in Nyasaland, taken on
nine consecutive days from Rat 2300.
ae ee | ay
Ft spusUBuaaacceEraGaEEEe
AA
IL PTE NES EE sea [EST
SI, HCGee om we
| el dC) Ne
SSCAG000e cose
The Zululand strain, 1913, has been carried on in horses and cattle for
some time in the laboratory at Pretoria, and may have varied somewhat in
morphology under these artificial conditions. It will be interesting to see
what change, if any, is induced by passage through G. morsitans. The three
following curves represent first, second and third passages :—
|
E
}| SSSR eee) eee
EEE ALE A}
||
g
Cuart 6.—Curve representing the Distribution, by Percentages, in respect to Length, of
500 Individuals of 7. brucei, Zululand Strain, 1913, after first passage through
G. morsitans, taken on nine consecutive days from Rat 2006.
ee ED
El [13] 14]15] 16] 17] 18]19]20|21[22 123 lz |25]26] 27]28]29 [30] 31] 52]33|54 [35]
PeREREPT CTT Lene
eee eee Se ECE Acer
mee een
EERE AEE EEEEEEEEEEE
SSs355 7S e eens 2 oso
P Gena aor 0 Ue ema
SOHN eS
Se cey a ae
(eal
AEE CEE
tS 4s
504 Sir D. Bruce and others. Description of a
Cuart 7.—Curve representing the Distribution, by Percentages, in respect to Length, of
500 Individuals of 7. brucei, Zululand Strain, 1913, after second passage through
G. morsitans, taken on nine consecutive days from Rat 2288.
[iy OES OSs 00 OP ee cron (6cl ac eee
[SI '13[14]15]16]17] 18 [19] 20]21 [22]23/24]25]26]27 [28 | 28/30)
10
Fil
Hee!
IoGa SOWERLOOO Joi
Percentage
rere
PEGE ae ee
VARESE R eR Aue
HARE ERSRSENERee
oe it Tif Alice TE as
Cuart 8.—Curve representing the Distribution, by Percentages, in respect to Length, of
500 Individuals of 7. brucez, Zululand Strain, 1913, after third passage through
G. morsitans, taken on nine consecutive days from Rat 2406.
che aes Seon aaa
Hj t ff}
ae eee
Bee enoLeesec
[es i TES SaShe
Strain of T. brucei from Zululand. 505
From this curve it will be seen that three passages through @. morsitans
has had little or no effect in changing the character of this trypanosome as
regards distribution of length. It has usually been thought that a trypano-
some kept under laboratory conditions, and without the opportunity of
passage through its invertebrate host, the tsetse fly, would tend to change
in morphology. These curves, on the other hand, show that a trypanosome,
after passage through horses and cattle for some years—exact time unknown
—is unchanged by three passages through its invertebrate host, G. morsitans.
Table 1V.—Distribution in respect to Length of 500 Individuals of 7. brucei,
Zululand Strain, 1913, after first passage through G. morsitans.
| In microns.
Average
| | | | | | length.
16. | 17.| 18.| 19. | 20. | Dil || Be, || BBE || BE)! eae || Be ie ee hee [eee 32.
| | i t t { |
| | | | | | | |
eee —| 1] 1 GF =| B Epo | 4) Bp} 2)—)-2 =) i ois
Bee tietecces = == I 3) — 4 3 ed eS ara le 1;/—|]— | 23°8
Beebe 2: ae || Tl Be SE Dela es Ey | ST Ae feet oe ieee —— ea opr
es... ee ae SO aware 3 eS) 1 | eo eee [eT | | ope
Be! c..- —j i] i if 2 6 3; 1| 4/—|/—]|—}] 1}/—|—|—|]—| 214
Pe ene = =) B= 3 6 4) 2} 2} 1)/—}/—|—j|—|—|;—;—)| 21
ote —|—|—j| 8 4| 4 rp Phat Ua Ee a 1g a ay |
Sites —|—| 3 1) 8}) 3} —J 2} 1] 2}—}-1)/-—}/—}]—|/—|—] 2210
ites. 2.0: —|/=—| 3| 383 sili a! 2)e2/—|—|—| 2)—)]- 2/—}—|—] 217
Bee 3s.. —|—!/ 2 2 a TT ae Ne! Hee MN aT eit 8 eA ie lee et | sal aco
see — | —} — 3 Sa Tht RN) a a a eee ae ahah Dar
ees —|—j; 1 Doe | Rel RV] (SRE ORE S41 gee el MW Ws Ug a (et Wren Fe VRE Wen
Baas otis > « —|— 2 4 iy, 1 4 — 2 30 74 | || fe 22 +4,
me, —|—! 1 Tee Til ear 2) 2] 5] 8| 2}—}—] 1] The || eae
ssnckb eee | 1 eer 2 Be SNR SUN al eae en Gy | eee eel ale ce
cca eee —| 2) 2 2 Mish res Th By A We Te ee oe! = Le
cochePppacey —j| 1] 8 Le ra a By) ey] ee | 21°8
ace ECCE ED —!|—] 2 Sy ne 79) Sie 1}; 5)/—/ 1);—) 1)—); —| —}] —] — 21°3
eS: —|—j)} 1 5 4| 5 Hy ae | ee le i Te | ee ee ee
Beco: yey a Tig Vc se Be By eh TW Tyee Be al ees | ee)
Be sce —;/—/] 1| 5 1 4 2) | Sy ee eet ae Bie
Bee ons. eA coor 4s | Tl A) I | |) ers
Pee aitacses —}|}— iL 2 Seed: P|) Oe DE | then le at 22-1
WS os —|—|— atel ZN teat?) Sone Ae a Zale AE ee eh ae | ee) ae
Beye ses oes =) 0 Si 5 Ari) Folie 1|—]| 2 SOM cae ee | Sane
| | | | | |
| | | | | |
cece TS 7 Bi ES er) m| 48/47/43 ao |a5/is| 1021) 6 | 2 2
{ |
+02 1°4)7-6|11°2/13°4)14-2| 9-6 94/86 809036) 42/10 0-4 0-4|
|
506
Sir D. Bruce and others.
Description of a
Table V.—Distribution in respect to Length of 500 Individuals of 7. brucei,
Zululand Strain, 1913, after second passage through G. morsitans.
In microns. mi
= verage
Animal. ih ae
13.| 14.| 15.| 16.| 17. | 18. | 19. | 20. | 21. | 22.| 28.| 24] 25.) 26.) 27-
Rat eateiee mete =/=/=—/— 1 I 5 | 4] 2 | 38.) ohhe dees ees one
an emo cee tt ed Sp Nee 4 3 ier eet) 7 — | LO)
Ooh Reale eS = |) | els 3 2 3 2) Vio 2) 2) Toe ee on 7
AR AT 3: Penh =/—/—)— il 3 4 5 4|.25),.0 | =) SS eo
ai a at as =/|/=—/—/— 2 2 9 3 Sy eS a OES
SR PONE == fs_ i} iL) = Bil 5 5 pl = | cee | —|—!] i9@
OR Anta Hae =| =| Wy 8 A 5 1 Oa Tn | eed a —|—] 1971
DS Be atk ne =/=| i)— 5 2 2 4 A) 1) = | = 2S eee
ol Sao Meee =/|—=|—/| al — 2 5 3 eer) | || — | 19m
MOLES. eck ee —=|/—]|/—) 2 il 3 3 5 3) |) 2) ee ee LG
2 SN Rae =|=—] i|}=— 2 3 5 5 pe] | ies
1 hee ob abe 3 =—/|—_/)|/—] i 3 4 it 3 4. | <2} 0) se a ee Oe
Se RS te =| 2B) ai & 4 3 2 2 pea ey
cod ae Fao i |=} a 2 4 6 35) = = |) Bh) Se ests
Dig ate ne =|) a} i 6 5 3 eels ff | aL
SH eo Oe =|/=/—!| 2 3 5 2 3 1) 22) 0) oc = Ss eon
Aiea ee seen a =—|—) =) — 2 il 7 5 Ne ie es pes ee ee |
Bo ie ae =/—|/=—/—! «2 3 4 4 Sy | | eas aaron
eee ET i ie eed | Toles 38 3 3 2 Bef od) a) ee ee a eon
i pl gs i =/|=] tf) = 2 5 5 | — | 6) 0 a |) ee eecoro)
PDE ARISE Nas =/S=)/—)—]! —}- 7 5 Sylkeae} al | yo) a aeons
Bt eR AcE =|)/=—]/=|= i. 2 4 5 CE ON Os | | Ra) a
aC FCA Ee aS = |/=|—|/—/| 2 2 2 il 4 | 46.) 37) 2a) 1 ee oe)
Page ares Myint a —/=—|/=]'=) i} 2 2 2 8.| 63. |) 2] 4g] ee od
PR REE ieee es sft Wh oe] ye 5 3 =| Aes} — | — |) 192
Motel autres i) 2) Si abe) eB | a Or | SS) Gr 1 S788) 20) BY] al 2
Percentages ...|0°2|/0°4|1°6|3°4|10°6/13-2|19-4|17-6|13-4|7°4 6°6|4-0|/1-0/0°8|0°4
507
| Average
€ ODF OPO GO COOAPAAOOOABHONAD
q Pel a el Sil IOS AS Salalas
o NANNAANA NANNANNANANAANANAAAAN
ea
es - = Secs eer a a a nN
Ri PA TEL AR ea A ear eae 2
5 me ra i g jo)
x ee a a ees jenl mail ethene (iif bazialt ball iz
. RORY Ry re ’ — =F : =i
ni NYE Tete PT ae te eT Tea TT Ee
S teem am lf fag rsa bn ek ea ee
24. | 25. |
23.
22.
|
!
fo 08
7-3
MW MMAMANMMIONATHADOAMAN MH HH tes
In microns.
21.
20.
AwWOOMNDNANRAHNA ANON A AN AN H10
OD OD OD AV ry 19 19 19 AI HO OD NI OD A I HOO A 10 10 19 FH
Ar ry rt I OD HD OD 0 a SH OOD NI rd NT OD 10 OD OD tt OD OD
Strain of T. brucei from Zululand.
17. 18. | 19.
Zululand Strain, 1913, after third passage through G. morsitans.
Table VI.—Distribution in respect to Length of 500 Individuals of 7. brucei,
pli ala th SSeS elle ei ate ale vare ian COSA
|
|
|
|
|
|
2°8 8°2/11°6/19-2 rise MOORE
eee a haa
Percentages 0°2
Rotalt 2402.22.
508 Sir D. Bruce and others. Description of a
Cuart 9.—Curve representing the Distribution, by Percentages, in respect to Length, of
2000 Individuals of 7. brucei, Zululand Strain, 1913, taken on nine consecutive
days from Rats 1829, 2006, 2288, and 2406.
DM (0 a
[ | espearsrrervn ot ra[oo Tot a eos [nev [oe sols [salsa ssa
a ||
EREERUSSSSRReeo
FECRGGGA Gg cOeecceo
EER ESSENSE
eT] a a EPH
As Shilston’s mule was in all probability infected from the wild game of
the Somkele district, in Zululand, it will be interesting to compare this curve
with that of the trypanosome causing disease in man, taken from the wild
game in Nyasaland.
Cuart 10.—Curve representing the Distribution, by Percentages, in respect to Length, of
2500 Individuals of the Trypanosome causing Disease in Man in Nyasaland, the
Wild-game Strain, taken on nine consecutive days from. Rats 847, 1220, 992, 849,
and 1022.
a Ea TC en ee Te]
[seeps iTS
Sa a a LL ae
3 Ken i sa a ea
} See
sell cee
oT hel Nu Sea a a Oe a
a edlasrantdtasstittcces
13
SSG
et a a a
oe ee
Strain of T. brucei from Zululand. 509
Table VII.—Percentage of Posterior-Nuclear Forms found among the Short
and Stumpy Varieties of 7. brucei, Zululand Strain, 1913.
Experiment | ; Percentage among short |
| bets tno! Lavan and stumpy forms.
|
1912 | |
| aie: PA caceace I Horse 30 |
ieee II is 65 |
— I Dog 5
dunes Zl. Il i Nil
1913. |
Hebets. ice: 1828 Rat 1
PGi 1828 : 37
Aa EONS 1828 | a 63
a 0 eee 1828 3 36
bod (ope lace ete 1828 3 32
ri GIS. BI. a4 1828 a 17
Caras eee 1828 is 54
|» 16......... 1828 is 45
Pie, 6 fk. 15286 || : 20
oS chee! Verret 1828 3 17
Mar. 24......... 2006 i 10
a UO ae 2006 if 54
ir Joh ol area 2006 ‘ 41
ie pe” aa 2006 is 18 |
at ee eesorbeee 2006 oo 38 |
gOS. Lh. 2006 if 32
pA PY yer esemce: 2006 - 69
hed ieee 2006 eo 74.
bet i Ortss kaa « 2006 Fe 63
Sees 2006 is i |
dilly? Al oeeeceee 2288 “7 Nil }
is See ae 2288 , 42
Fe eOecentenes 2288 op 20
» 24 2288 f 35
A oC nacre 2288 16
opp WS eeebanpe 2288 ; 29
i ree 2288 6 | 43
Bt NNR Re! 2288 ef 52
SRO ee 2288 5 49 |
ey ROM Repebe mae 2288 vs 61
a eet ee | I Mouse | 5 |
ASV EAD Camere tectier 37 8
These two curves are undoubtedly much alike, and as the wild-game strain
in Nyasaland is supposed to be identical with the human strain, then it
might be said that 7. brucei, Zululand strain, 1913, is also identical with the
trypanosome causing disease in man in Nyasaland. Others will say that the
Zululand trypanosome and the Nyasaland wild-game strain are both 7. brucei,
but that this does not prove that 7. brucei is identical with the human strain.
No, but if it is shown that 7. brucei, Zululand, is absolutely identical in
morphology with this Nyasaland human strain, that it also has exactly the
same disease-producing power on the various experimental animals, this will
VOL. LXXXVII.—B. 2 Rk
510 Description of a Strain of T. brucei from Zululand.
go a good long way to make the ordinary unprejudiced man chary of exposing
himself too carelessly to the so-called harmless nagana. His serum may save
him as a rule, but there may come a time when—his resistance being lowered
either by fatigue, or some other cause—the trypanosome may gain a footing,
and then his belief in the written word of the text-book will receive a rude
shock.
Breadth.—The long and slender average 2°76 microns in breadth, the
intermediate 3:25, and the short and stumpy 3°53. This measurement was
made across the broadest part of the trypanosome and includes the
undulating membrane. Most previous measurements of breadth have not
included this. ©
In regard to the shape of this trypanosome, contents of cell, nucleus
micronucleus, undulating membrane, and flagellum, it is not proposed to
describe these characters separately for this strain as was done in the case of
the trypanosome causing disease in man in Nyasaland. Suffice it to say that,
after the most careful comparison, no difference whatever can be made out in
the morphology of the two trypanosomes. At the end of this paper three
plates are given, one representing the short and stumpy, another the inter-
mediate, and a third the long and slender forms. If these plates are
compared with those given in previous papers* it will be seen that in
morphology the Nyasaland and Zululand trypanosomes are identical.
CONCLUSIONS.
1. The trypanosome described in this paper under the name of the
“Zululand strain, 1913,” is the same species as that discovered by Bruce in
Zululand in 1894; reported on by Kanthack, Durham and Blandford in
1898 ; and named 7’. brucei by Plimmer and Bradford in 1899.
2. As regards its morphology, this trypanosome is absolutely identical with
the trypanosome causing disease in man in Nyasaland, the 7. rhodesiense of
Stephens and Fantham.
DESCRIPTION OF PLATES.
PuateE 21.
Trypanosoma brucei, Zululand strain, 1913, short and stumpy forms. x 2000.
PLATE 22,
Trypanosoma brucei, Zululand strain, 1913, intermediate forms. x 2000.
PLATE 23.
Trypanosoma brucei, Zululand strain, 1913, long and slender forms. x 2000.
* ‘Roy. Soc. Proc.,’ B, vol. 85, p. 483 (1912), and vol. 87, p. 35 (1913).
Bruce. det.
Shar
& Stam py.
Poy, Soc. Proc. B.vol.8f, Pl. 27.
avid Bruce & others. ae Roy. Soe Proc. B. vol.87 Pb.22.
Intermedtimte.
FE. Bruce, det.
| Sir David Bruce & others. hoy Soc. Proc. B. val. 87, PLAS. *
Ly ong & Slender.
ME Bruce, del.
dll
Description of a Strain of Trypanosoma brucei from Zululand.
Part Il.—Susceptibility of Animals.
By Surgeon-General Sir Davip Brucs, C.B., F.R.S., A.M.S.; Major A. E.
Hamerton, D.S.0., and Captain D. P. Warson, R.A.M.C.; and Lady
Bruce, R.R.C. (Scientific Commission of the Royal Society, Nyasaland,
1912-14.)
(Received February 24,—Read March 26, 1914.)
INTRODUCTION.
In the foregoing paper the morphology of this trypanosome was described,
and the conclusion arrived at that it is identical, as regards shape, size and
general appearance, with the trypanosome causing disease in man in
Nyasaland, the Trypanosoma rhodesiense of Stephens and Fantham.
This paper describes the action on animals of the Zululand trypanosome,
and it is compared in this regard with the Nyasaland species.
SUSCEPTIBILITY OF ANIMALS TO T. BRUCEI, ZULULAND STRAIN, 1913.
Table I.
No. of c Incubation,| Duration,
Date. expt. Source of virus. in days. in days.* Remarks.
Horse.
1895.
= Gif) | 1Diayy er ceoacbocscneacoosn56 3 35 “Very old animal; typical
| Nagana.”’—Shilston.
Sept. 27...; 212 | Natural infection ...... EB 30 Zululand, 1896, Bruce.
- 29... 235 i Syl meaaeine 6 49 » ” »
| —__—_—.-§ |
Average...... = 38 0
Ox.
1913
— 22) MAD OR Awe cusecannacsecades 6 a “Still alive after 90 days.” —
Shilston.
Telos MSecolf TB |) Teens 88) coucocosoeaccnoos = = Never showed trypanosomes.
See | LOA PERS OO ee sees cae 37 = Still alive after 316 days.
“3 dhe) SIGIR. |) Ese CBE ane 35 310 Died of 7. bruce.
July 22...) 23804 | Dog 2281.................. a = Never showed trypanosomes.
5 DPA CRUE Cn bbe il aie ie! — -- cf ip
NORIO || eT) eae = — , 9
Sheep.
— CD OCEAN Eel aecttsect claves 12 _ “Still alive after 90 days.” —
Shilston.
* Duration includes the days of incubation ; it dates from day of infection.
Dy Te Py
512 Sir D. Bruce and others. Description of a
Table I—continuwed.
Date. ae Source of virus. aaa pans Remarks.
Goat.
Feb. 12...) 1887 | Rats 1832 and 1838 ... = — Never showed trypanosomes.
» 12...{ 1888 », 1832 and 1838...) — = ¥ i
» 12...) 1889 », 1832 and 1888 ... 15 109 Died of 7. brucei.
x) Woo) ANSI ,, 1832 and 1838 ... = = Never showed trypanosomes.
Mar. 15...) 1890 | Guinea-pigs 1840 and 12 | 45 Died of 7. brucet.
1843
July 16...| 2290 | Dog 2254................6 26 | 39 50 x
1G il DBO ata Memeo oa eee cee ee ene 36 | 116 i ‘s
Average...... 22:2 Clef Pe
Monkey
Feb. 3...) 1833 | Rabbit 1880 ............ f 8 Died of 7. brucei.
Pesuelwdese ab B Ogee Senne Hi 15 i i
yy Bena|| Ufc B15) Be OBO) treaeceeneee 7 14 % rf
5 Sash HERG OY. < SRO Hk ee 7 49 . -
8. a alSaa 3 AR80) Eee ee z 16 ‘ x
» 17...) 1970 | Laboratory-bred flies... — 17 ~ i
July 16...) 2292 | Dog 2254.................. f 5 50 5 5
Fy, LOsee|) 2298 alt 2204... crn. cree 5 65 _ ie
| eee a
| Average...... 6°4 29 -2
Dog.
Feb. 14...| 1904 | Monkey 1835 ............ | 6 26 Died of 7. brucei.
4) 1) 1905 a amo een eas. | 6 17 ss &
» 14...| 1906 WRG B Lee ieee 10 18 i i
5 1404) 1907," |
—= 158 Horse Bi henrnea ieeaace 3 27 »
— 165 Rabbit 158 ............... 5 27 »
Jan. 13..., 1897 | Pretoria strain ......... P 36 Died of T. brucet.
” Wye 1830 | ” 99 te tee ee P 35 0 ”
Average......| 7:0 | 82°7
* Duration includes the days of incubation; it dates from day of infection.
Strain of T. brucei from Zululand.
Table I—continued.
513
Wate No. of Soustoretisus Incubation, | Duration
expt. , in days. in days.*
Guinea-pig.
Feb. 3...| 1840 | Rabbit 1830 ............ 31 50
Pers. 1841 PBI SSO ees 35 50
Mee tae NWOT igsghe MeO: an =
5) <=] 1843" | oy AUEEIO pegecsocoaas 21 59
MRS NSA Psy) TSBO occ cues 14 37
» 138..., 1895 | Pretoria strain ......... = =
los Soo of Fruits yyeseedoes —_ =
Mar. 28...) 1842 | Monkey 1970............ 10 27
» 28...) 1895 LG 170s 20 46
» 28...) 1896 ROMNEY (Oipeiccnke ve 20 30
Matyas 2186 ||. (17,,0)4 1970). nee. ae =
fe-29,.)|" 2136 SMBS TOM. 4 15
June 13... 2225 STO Oe 3 34
July 16...| 2296 | Dog 2254......0....2..0:.. 47 89
Average...... 20°5 43 °7
Rat.
Feb. 2...| 1828 | Rabbit 1830 ............ 8 19
a bool) LIBPAD ss SOO) cers ease 6 58
pees | 1g82 eet Ga0 PUTO 7 46
Ry eS). 1888 Pr TGSOF Waa 10 13
pote) 1839 ie ATEN oe) 7 22
eae 1902 SHR LOON eater 17 31
Ores) LOGE Guinea-pig 1844 ...... 8 23
Marre Sens L993e | Rab L8daeecn cess ceere 5 13
», 16...) 1994 pote SB YP anaeeensecreocboe 5 10
ppeelome elo 9e pal dnsBYA ndopocepooascbac 5) 30
», 19...) 2006 | Monkey 1970 ............ 5 24
April 4...) 2065 Syeda secede see 7 30
PEN aA eI078i— | Rats2060..00 9.006). ck 4 17
May 13...) 2135 | Monkey 1970............ 6 33
5) Ld...) 2137 | Goat 1889 7... 6 18
» 13...| 2188 mF dustsl’)) aepcochonene pee 6 17
oy WQecel| PHYS) iets PAB 5) 45 sooceaconeadose 11 26
July 16...) 2288 | Dogi2254,..)...........+:- 5 33
MTG P280rN Onna see ee 5 44,
Sept. 2..., 2406 Baht LOO limemaaeen tenes te | 6 24;
MI GHHe 2402, te Rate 2406..5,04ee.: 05: | 6 34,
Oct. 20...) 2423 ROAR nein aeciiecs | 8 39
Nov. 28...) 2442 She SAO cae ae ceptiechieels | 4 16
eae Sa
Average...... 6°8 27-0
Remarks.
Died of 7. brucei.
> ?
Never showed trypanosomes.
Died of 7. brucei.
” 2
Never showed trypanosomes.
2A ” se 2 5
Reinjected; died of 7. brucei.
” ” ”
Never showed trypanosomes.
Reinjected ; died of 7. brucet.
Died of 7. brucei.
»” a”
| Died of 7. bruce.
* Duration includes the days of incubation; it dates from day of infection.
Disease set wp in Various Animals by T. brucei, Zululand Strain, 1913.
Horse.—The Commission had no opportunity of studying this strain in the
horse, but Mr. Shilston states that one horse inoculated by him at Pieter-
maritzburg died in 35 days with typical symptoms of Nagana.
Ox.—Six oxen were inoculated, but only two of these at any time showed
trypanosomes in their blood. One of these died after 310 days, while the
514 Sir D. Bruce and others. Description of a
other is still alive at the end of a year. This animal has evidently recovered,
as it appears sleek and healthy. The action of the Zululand strain is
therefore the same as that of the trypanosome causing disease in man in
Nyasaland: neither of them show any marked power of producing serious
disease in cattle. i
Goat.—Seven goats were inoculated with this strain. Four died, on an
average, in 77:2 days(45 to116). The remaining three proved refractory. No
cedema of face or corneal opacity was noted in any of the goats. The
Zululand strain seems to have less action on goats than the Nyasaland
trypanosome, but the number of experiments is small. In the latter the
duration of the disease was 41°8 days (19 to 72).
Sheep.—No experiments were made with these animals in Nyasaland as it
was found impossible to procure them from the natives.
Monkey.—Fight monkeys died, on an average, in 29°2 days (8 to 65). The
trypanosomes were always present in the blood, sometimes in enormous
numbers. In no case was cedema of the face or corneal opacity noted. After
death, enlargement of the spleen and liver, gelatinous infiltration at the base
of the heart, and hemorrhages in the epicardium were found.
Dog.—Seventeen dogs were inoculated. All died, on an average, in
185 days (12 to 26). In eight dogs blindness caused by opacity of the
cornea was a prominent symptom, and in two swellings of the limbs were
observed.
Rabbit—As only two rabbits were available at Kasu, six experiments
reported by Mr. Shilston are added. Eight rabbits died, on an average, in
32°7 days (27 to 39). The course of the disease in the Kasu rabbits was the
same as that described in a former paper™ as being typical of Nagana.
Guinea-pig—tThis animal is less affected by the disease than the rabbit.
Ten were used; all took the disease and died, but four required to be
inoculated more than once. They died, on an average, in 43°7 days
(15 to 89). No prominent symptoms, such as are seen in the rabbit, occur
in the guinea-pig.
Rat.—Twenty-three were inoculated and died, on an average, in 27 days
(10 to 58), with their blood swarming with trypanosomes and their spleens
enormously enlarged.
* “The Trypanosome causing Disease in Man in Nyasaland.—Susceptibility of Animals
o the Human Strain,” ‘ Roy. Soc. Proc.,’ B, vol. 87 (1918). .
Strain of T. brucei from Zululand.
515
Table I1—The Average Duration, in Days, of the Disease in Various Animals
eaused by 7. brucei, Zululand Strain, 1913.
| |
Eigse Ox. | Goat. | Monkey. | Dog.| Rabbit. | Guinea-pig. | White rat.
:
Average duration,| 38 | 310 | 77 29 18 33 44, 27
in days |
|
Number of animals 3 1 a 8 17 8 10 23
employed
Compare this with the following table :—
Table I1I.—The Average Duration of Life, in Days, of Various Animals
Infected with the Human Strain of the Trypanosome causing Disease in
Man in Nyasaland.
Goat and
Horse.| Ox. Monkey. | Dog. | Rabbit. | 12°") White rat.
sheep. | | pig. |
Average duration, 9% 134 42 26 34 28 67 30
in days
Number of animals 0 1 29 20 25 U 15 2L
employed
Table [V.—The Percentages of Recoveries in Various Animals Infected with
T. brucei, Zululand Strain, 1913.
Compare this with the following table :—
| |
tiene Ox. al Monkey. Dog. | Rabbit. | Guinea-pig. | White rat.
|
Percentages) -...-4.-- 0 83 0 | 0 0 0 0) 0
Number of animals| 3 6 4 | 8 17 8 10 23
employed
Table V.—The Percentages of Recoveries in Various Animals Infected with
the Trypanosome causing Disease in Man in Nyasaland.
|
Percentages
Number of animals
employed
|
Horse.| Ox. Neca “un
| sheep.
(0) 80 (0)
0 5 29
Monkey.
| ayers, || Tenses, | GR
| Pig:
0 0 0
25 7 15
White rat. |
21
516 Sir D. Bruce and others. Trypanosome
CONCLUSION.
The pathogenic action of 7. brucei, Zululand strain, 1913, on various
animals is so similar, not only in regard to the symptoms during life
but also in the post-mortem appearances and rate of mortality, to that of the
trypanosome causing disease in man in Nyasaland, that it affords another
proof that these two trypanosomes are identical.
The Trypanosome causing Disease in Man om Nyasaland.
Part ITI.— Development in Glossina morsitans.
By Surgeon-General Sir Davin Bruce, C.B., F.R.S., A.M.S.; Major A. E.
HamertTon, D.S.0., and Captain D. P. Warson, R.A.M.C.; and
Lady Bruce, R.R.C. (Scientific Commission of the Royal Society,
Nyasaland, 1912-14.)
(Received March 17,—Read March 26, 1914.)
[PLatE 24.]
INTRODUCTION.
In previous papers* the morphology of this trypanosome and the suscep-
tibility of various animals to its pathogenic action have been described. In
this is given an account of its development in Glossina morsitans.
In Uganda the study of the development of Trypanosoma gambiense in
G. palpalis was much assisted by the circumstance that large numbers
of laboratory-bred tsetse flies were available. This was due to the fact
that the pup of G. palpalis could be collected on the lake-shore in prac-
tically unlimited numbers. It is quite otherwise with G. morsitans. It has
been found impossible to find the pupe of this species in any numbers, so
that all laboratory-bred G. morsitans have had to be hatched out of pup
obtained from captive flies, a slow and laborious process. ‘The flies are
caught some 20 to 30 miles from the laboratory and brought up to Kasu camp
by a native on a bicycle. This kills a large number of the flies. Moreover,
the climatic conditions at the camp are not always favourable for breeding
and hatching out. This was remedied to some extent by establishing a
breeding station down in the low-country, but as this had to be left in the
tharge of natives the results were not always very satisfactory.
* “Roy. Soc. Proc.,’ B, vol. 85 (1912), and B, vols. 86 and 87 (1913).
causing Disease in Man in Nyasaland. Ly
The study of the development of this trypanosome in G. morsitans has
therefore been rendered difficult by the small number of laboratory-bred
tsetse flies which could be obtained. Over and above that, flies bred from
captive flies are not so strong and healthy as those hatched out from wild
pupe.
An attempt was made to use wild flies by feeding batches of about 20 on
healthy animals and picking out those cages which did not give rise to
infection. But this is at best a roundabout and clumsy method, as it can
never be certain, although every care is taken, that only clean flies are
being dealt with.
THE DEVELOPMENT OF THE TRYPANOSOME CAUSING DISEASE IN MAN IN
NYASALAND IN G. MORSITANS.
Eleven experiments were carried out with laboratory-bred flies. Three
were positive and eight negative.
Five experiments were also carried out with wild flies, as no lJaboratory-
bred flies were available. All were positive.
Tables I and II show these 16 experiments: the number of flies used; the
number of infected flies found on dissection; and the number of days which
elapsed before the flies became infective. As each fly died it was dissected
and the result noted. As will be seen from Table I, several infected flies
were found in the negative experiments. This probably means that the
flies were only infected, not infective. The number of days before a fly
becomes infective is arrived at by deducting seven days from the number of
days which elapsed between the first infected teed of the flies and the
Table IL—Laboratory-bred Flies.
| |
| | : |
| No. of | Experiment | . No. of days Temperature
Date. | Expt.| flies | sonitive or | meet wees before flies at aes flies
| used, negative. | tes Tounc’. | became infective. kept.
|
1912. | |
May 22 | 563; 18 — 0
June 13 668 22 _ 1
July 15 | 879; 32 - a
» 29|1003| 28 e 2 31 | 84°F. (29°C.)
Aug. 17| 1072} 27 = 3
Oct 23 | 1494 22 — 3
Nov. 6] 1560 | 19 _ 0
Dec. 13 | 1686 | 24 _ 2
» 28/1710} 30 = 0
» 80] 1723 | 35 + 3 14 84°F. (29° C.)
1913.
Aug. 31 | 2405 | 30 + 4 23 84° F, (29° C.)
| |
! |
518 Sir D. Bruce and others. Trypanosome
appearance of trypanosomes in the blood of the experimental animal. Seven
days is put down as the average number of days between the infection of the
animal and the appearance of the trypanosomes in its blood—the incubation
period. It is probably a day or two shorter.
The number of flies used in each experiment was small, due to the difficulty
of obtaining laboratory-bred flies. They were kept during the experiment in
the incubator at a temperature of 84° F. (29° C.).
‘In Experiment 1723 the number of days which elapsed before the flies.
became infective is only 14. This number is obtained, as mentioned above,
by deducting seven days for the incubation period, but this may have been
a day or two less. The flies were kept at an evenly warm temperature, which
would tend materially to shorten the period of development. Still, 14 days
seems a short time to elapse between the first feed on the infected animal
and the appearance of an infective fly in the cage.
Two hundred and eighty-seven laboratory-bred flies were used and 25
infected flies were found—8’7 per cent.
Table II].—Wild Flies.
|
No. of Experiment 5 No. of days Temperature |
Date. | Expt.| flies © ponte or gage infected before flies | at ah flies
- ies found. : :
used. negative. became infective. | kept.
1912.
Dec. 11 | 1680 80 + 8 | 18 84° F. (29°C.)
» 18 | 1688 40 + 6 3 84° F. (29° C.)
SO VHS"| 1705) | 045 + 7 1 84°F, (29°C.) |
1913.
Jan. 9 | 1748 70 + 1 25 84° F. (29° C.)
14) 1729) 20 + 1 30 84° F. (29°C.)
Experiments 1688 and 1705 are evidently cases of infection by naturally-
infected wild flies which had escaped detection. They are included in the
table as they both show invasion of the salivary glands and so help to throw
light on the mode of development of this trypanosome in G. morsitans. The
other three pass through an interval of 18, 25, and 30 days before the cages
became infective. These are probably cases where there was no naturally-
infected fly in the cage, and these periods therefore represent the usual length
of time required for the cycle of development of this trypanosome to take
place in G. morsitans. The wild flies were also kept in the mcubator at
a temperature of 84° F.
Two hundred and fifty-five flies were used and 23 infected flies were found
—9 per cent.
causing Disease in Man in Nyasaland. 5L9
Details of the Hight Positive Experiments.
The following table gives the details of the eight positive experiments :—
Table III.
Expt. Day of expt. Procedure. Remarks.
1003 1-2 Flies fed on infected dog. Trypanosomes appeared in
3 Starved. blood of Monkey 1023 on
4-4] Fed on clean Monkey 1023. the 38th day.
1723 1-4 Flies fed on infected dog. Trypanosomes appeared in
5 Starved. blood of Monkey 1733 on
6-22 | Fed on clean Monkey 1733. the 21st day.
2405 1-6 Elies fed on infected monkey. | Trypanosomes appeared in
7 Starved. blood of Monkey 2410 on
8-32 Fed on clean Monkey 2410. the 30th day.
1680 1-2 Flies fed on infected dog. Trypanosomes appeared in
3 Starved. blood of Dog 1708 on the
4-22 | Fed on clean Dog 1708. 25th day.
1688 1-2 | Flies fed on infected monkey. | Trypanosomes appeared in
3 | Starved. blood of Monkey 1699 on
4-12 Fed on clean Monkey 1699. the 10th day.
1705 1-2 | Flies fed on infected monkey. | Trypanosomes appeared in
3 | Starved. blood of Monkey 1707 on
4-9 Fed on clean Monkey 1707. the 8th day.
1748 1-2 | Flies fed on infected monkey. | Trypanosomes appeared in
3 Starved. blood of Monkey 1845 on
4-30 . | Fed on clean Monkey 1845. the 32nd day.
1729 1-2 | Flies fed on infected dog. Trypanosomes appeared in
3 | Starved. blood of Dog 1767 on the
4-38 Fed on clean Dog 1767. 37th day.
Omitting Experiments 1688 and 1705, it would appear from the remaining
six experiments that an average period of 24 days is required to complete the
cycle of development of the trypanosome causing disease in man in Nyasaland
in G. morsitans, the flies being kept at a temperature of 84° F.
Details of the Hight Negative Expervments.
The following table shows the method of procedure in carrying out the
eight negative experiments :—
520 Sir D. Bruce and others. Trypanosome
Table LV.
Expt. Day of expt. Procedure. Remarks.
563 1-3 Flies fed on infected monkey. | All flies negative on dissec-
4 Starved. | tion.
5-52 Fed on clean Monkey 594. |
668 1-2 | Flies fed on infected dog. | One infected fly found on
3 | Starved. the 42nd day.
4-63 Fed on clean Dog 699. |
|
879 1-2 | Flies fed on infected monkey. | Seven infected flies found.
3 | Starved.
4-32 | Fed on clean Monkey 910. |
33-63 Fed on clean Monkey 1073. |
1072 1-3 | Flies fed on infected dog. | Three infected flies found.
4. Starved. |
5-54 Fed on clean Dog 1148.
1494. 1-3 | Flies fed on infected monkey. | Three infected flies found.
| 4-5 | Starved.
6-44, Fed on clean Monkey 1514.
|
1560 1-3 | Flies fed on infected monkey. | All flies negative on dissec-
4 | Starved. tion.
5-37 | Fed on clean Monkey 1581. |
1686 | 1-4 | Flies fed on infected monkey. | Two infected flies found.
| 5 Starved.
| 6-43 Fed on clean Monkey 1704.
1710 | 1 Flies fed on infected dog. All flies negative on dissec-
2 Starved. | tion.
3-47 Fed on clean Monkey 1718.
RESULT OF THE DISSECTION OF THE INFECTED FLIES.
All the flies dying during the progress of these experiments were dissected.
In the three positive experiments with the laboratory-bred flies nine infected
flies were found. The following table gives the results of the dissection of
_ these nine flies. The second column gives the number of days which elapsed
between the fly’s first infected feed and its death and dissection. In the
third column the labial cavity and hypopharynx are included under
“Proboscis.” At the time these experiments were made no attempt was
made to distinguish between the two parts, as has been done lately in the case
of 7. simiw.* When the proboscis is marked positive, as in Table VI, it may
be that the trypanosomes are contained in the labial cavity or the hypo-
pharynx, or both.
In the development of 7. gambiense in G. palpalis trypanosomes were never
* “Roy. Soc. Proc.,’ B, vol. 87, p. 59 (1918).
causing Disease in Man in Nyasaland. 521
noted as occurring in the proboscis.* In this species they are noted on
several occasions as occurring in this position, but only in the>wild-fly
experiments, not in the laboratory bred. It seems natural to expect that if
the salivary glands are swarming with trypanosomes that some of them will
sometimes appear in the hypopharynx and, moreover, in the wild flies some of
the infections of the proboscis are no doubt due to 7. pecorum, T. sume or
T. capre, all of which develop in the proboscis.
Table V.—Laboratory-bred Flies. Result of the Dissection of the Infected
Flies found in the Positive Experiments.
Expt. aa Proboscis. a Fore-gut. | Mid-gut. | Hind-gut. a
|
1008 | 33 = a =
1003 39 = + ?
1723 30 — ++ + + ++ ++ =
1723 30 - ++ ++ ++ ++ —
1723 48 = = = = = =
2405 32 | + =
2405 33 — ++ ++ | ++ ++ ++
2405 33 = = + | + + =
2405 | 33 = = + cE + =
In Experiment 1003, two infected flies were found. The first had only a
gut infection and, unfortunately, it was found impossible to dissect out the
salivary glands of the second. Neither had an infection of the proboscis.
In Experiment 1723, three infected flies were found. The first and second had
the alimentary tract swarming with flagellates, but none in the salivary glands.
The third was found on dissection to be free from trypanosomes throughout.
This is curious because this fly had been isolated in a glass tube as an
infective fly, and had, when used alone on a rat and rabbit, infected both
these animals. The fly remained alive in the tube for 13 days, and the only
explanation that can be given is that in this case the trypanosomes disappeared
absolutely from the fly some few days before its death. This was the first
time this had been observed to take place, and it was thought to be a
remarkable phenomenon and difficult to credit, until another example of the
same kind was observed. It must, therefore, be held as probable that an
infective fly, with presumably both salivary glands and alimentary tract
swarming with trypanosomes, can lose all these flagellates and become non-
infective.
In Experiment 2405, four infected flies were found. Three of these were
infections limited to the gut. The fourth was a good example of a salivary-
* TIbid., B, vol. 82 (1910).
522 Sir D. Bruce and others. Trypanvsome
gland infection. The glands were swarming with trypanosomes, and a portion
of one of them injected under the skin of Rat 2417 gave rise to infection.
Table VI.—Wild Flies. Result of the Dissection of the Infected Flies found
in the Positive Experiments.
Expt. a | Proboscis. eae Fore-gut. | Mid-gut. | Hind-gut. mee
1680 5 - —_ + | -
1680 19 =
1680 32 = ++ ++ ++ ++ ++
1680 32 — — + - + _
1680 33 - ++ ++ ++ ++ _
1680 33 — = + —
1680 33 - — ++ ++ ++ -
1680 33 _ — + + — —-
1688 10 — + _
1688 10 — _ — + —
1688 11 _ —_ + _
1688 13 — + —
1688 15 - ++ ++ ++ ++ ++
1688 15 + — + -
1705 8 + + + + -
1705 8 — + + + -
1705 10 _ — + —
1705 11 - - -
1705 12 + + + + + ++
1705 26 — — ++ ++ ++ ++
1705 33 — + ++ ++ ++ ++
1748 31 — - ++ ++ ++ ++
- 1729 48 + - + + ++
In Experiment 1680, eight flies were found to be infected. In seven the
flagellates were confined to the alimentary tract. The eighth had a well-
marked invasion of the salivary glands. In this case trypanosomes were also
seen in the proboscis, but whether in the labial cavity or the hypopharynx is
not specified.
In Experiment 1688, six flies were found to contain trypanosomes in the
alimentary canal. In one of these there was also infection of the salivary
glands, which were crowded with trypanosomes. This fly must have been
naturally infected when caught, as sufficient time had not elapsed since the
infected feed to allow of time for development to take place. The flagellates
contained in the salivary glands injected into Rat 1721 gave rise to infection.
In Experiment 1705, seven infected flies were found. Three of these had
the salivary glands invaded. One of these, the fifth, must also have been a
naturally-infected wild fly.
In Experiment 1748, only one infected fly was found. It had a copious
infection of the salivary glands, a portion of which injected into Rat 1852
gave a positive result.
causing Disease in Man in Nyasaland. 523
In the last Experiment, 1729, there was also only one infected fly found.
The salivary glands were swarming with trypanosomes.
The next table gives the result of the dissection of the infected flies found
in the experiments which remained negative.
In the negative Experiments 563, 1560, and 1710, none of the flies were
found to be infected with trypanosomes in any part (see Table I). These
experiments are therefore omitted from this table.
Table VII.—Laboratory-bred Flies. Result of the Dissection of the Infected
Flies found in the Negative Experiments.
Time, 5 Proventri- | Fore- Mid- Hind- Procto- | Salivar
Beth days. EHONEORE culus. gut. gut. gut. deeum. giande
fal ka
668 42 — + + + _
879 0 — - + + + =
879 8 — + ++ —
879 9 = + + - =
879 11 - +
879 24 — + + + + — -
879 28 — ++ ++ ++ t+ =
879 40 — ++ ++ ++ + =
1072 7 _ = + + + =
1072 10 - + + +
1072 38 — ++ ++ ++ ++ ++
1494 7 — ++ - — — —
1494 17 — + + - =
1494 31 — + + =
1686 8 _ _ ++ ++ + =
1686 26 < = + + + =
From these negative experiments it will be seen that only in one fly did an
infection of salivary glands occur. Why this fly did not infect the animal it
fed on is impossible to say.
THE METHODS USED IN THE EXAMINATION OF THE FLIES.
The flies were dissected as described in a previous paper.* As each fly in
a cage died it was dissected, and the result, as regards the presence of
trypanosomes in the alimentary tract and salivary glands, recorded. Fixed
and stained preparations were then made from the various parts and numerous
drawings of the various types of trypanosomes encountered were made. The
method described in a previous papert of isolating infective flies and inducing
them to salivate on clean cover-glasses was also made use of. Thisisa useful,
simple and practical method, as it demonstrates clearly the type ot trypano-
some thrown out from the tip of the proboscis when the fly feeds.
* ‘Roy. Soc. Proc.,’. B, vol. 83, p. 513 (1911).
+ Ibid., B, vol. 87, p. 63 (1913),
524 Sir D. Bruce and others. Trypanosome
THe TRYPANOSOMES FOUND IN THE ALIMENTARY TRACT.
In this species of trypanosome the developmental changes which take place
in the intestine of G. morsitans are similar to those already described as.
occurring in the development of Z. gambiense in G. palpalis.* The latter
development has also been worked out very fully and completely by others.+
It is therefore unnecessary here to do more than refer to these previous
descriptions as being equally applicable to the species under consideration.
In this species of trypanosome also, as in 7’. gambiense, it is only a small
percentage of the flies fed on an infected animal which become infected. In
one series of 7. gambiense this was 8 per cent.t In this species the
experiments with laboratory-bred flies was 8°7 per cent., with wild flies 9 per
cent. Just asin 7. gambiense, the development takes place in the alimentary
tract and salivary glands and not in the proboscis.
THE TRYPANOSOMES FOUND IN THE SALIVARY GLANDS.
In the trypanosome causing disease in man in Nyasaland, as in 7. gambiense,
the crux of the whole matter is the invasion of the salivary glands. After a
certain number of days—in this species from 14 to 31—the trypanosomes
reach the salivary glands and the fly becomes infective.
Plate 24, figs. 3-28, represent the various stages in the development of this
trypanosome in the salivary glands. Figs. 1 and 2 are trypanosomes from the
proventriculus; these represent the dominant intestinal type, from which the
salivary-gland types arise. It is still a matter of speculation as to how they
gain access to the glands, but as described in a former paper,§ there is no
doubt they are often thrown forward into the proboscis during or, just in the
act of feeding, and may, under these conditions, be drawn into the hypo-
pharynx and so reach their destination. These proventricular forms, however,
have never been actually seen by the Commission in the hypopharynx.
Figs. 3-11 are forms found in the salivary glands. Many of these are
crithidial in type and occur in numbers. Figs. 12-14 are what appear to
be encysted forms. Figs. 16-21 are “blood forms” and occurred in large
numbers in the same preparation as the crithidial type shown in figs. 3-8.
Figs. 22-28 are “ blood forms” which were thrown out on to a cover-glass by
a living infective fly. The preparation was beautifully clear, each individual
trypanosome standing out distinctly. Fig. 23 is from the same preparation
* Tbid., B, vol. 83, p. 515 (1911).
+ Muriel Robertson, M.A., ‘Phil. Trans.,’ B, vol. 203 (1913).
t ‘Roy. Soc. Proc.,’ B, vol. 83, p. 514 (1911).
§ Jbid., B, vol. 87, p. 65 (1913).
i al,
ee
ae
SMe David Bruce & others Roy Sve. Proe. B. COl EF LL. 24
1S
g
16 17 7§
LO
Brypeanosome CAUsieg Disease ut Mar ur Nyasaland.
Developneent We Glosstita morsuarns.
M. FE. Bruce,dct X 2000
causing Disease in Man in Nyasaland. 525
and has the appearance of a small bunch or clump of “blood forms” in the
act of breaking apart.
CONCLUSIONS.
1. The trypanosome causing disease in man in Nyasaland belongs to the
same group as 7’. gambiense, the development taking place in the alimentary
tract and salivary glands, not in the proboscis, of the fly.
2. The percentage of flies which become infected is the same as in
T. gambiense, 8 per cent.
3. The percentage of flies which become infective is about 1 per cent.
4, The length of time which elapses before a fly becomes infective varies
from 14 to 31 days, average 25 days.
5. The infective type of trypanosome in the salivary glands—corresponding
to the final stage of the cycle of development—is similar to the short and
stumpy form found in the blood of the vertebrate host.
DESCRIPTION OF PLATE.
Figs. 1-2.—Trypanosomes from proventriculus. These represent the dominant intestinal
type.
Figs. 3-8.—Trypanosomes taken from a preparation of the salivary gland of an infective
fly. Many of these are crithidial in type, e.g., figs. 6, 7, and 8.
Figs. 9-15.—Other forms seen in the salivary glands. Figs. 12-14 have the appearance
of being encysted.
Figs. 16-21.—The fully developed “blood forms.” Without these the fly is non-infective.
These were drawn from the same preparation as figs. 3-8.
Figs. 22-28.—Trypanosomes ejected by a living infective @. morsitans on attempting to
feed through a cover-glass. Fully developed “blood forms.”
Stained Giemsa. x 2000.
VOL, LXXXVII.—B.
bo
RQ
526
Description of a Strain of Trypanosoma brucei from Zululand.
Part II].—Development in Glossina morsitans,.
By Surgeon-General Sir Davin Brucsz, C.B., F.R.S., A.M.S.; Major A. FE.
Hamerton, D.S.0., and Captain D, P. Watson, R.A.M.C.; and Lady
Bruce, R.R.C. (Scientific Commission of the Royal Society, Nyasaland
1912-14.)
>
(Received March 17,—Read March 26, 1914.)
[Puate 25.]
INTRODUCTION.
In previous papers™ the morphology of this trypanosome and its action on
animals were described. The chief object of this paper is to compare the
development of this species of trypanosome with that of the trypanosome
causing disease in man in Nyasaland—the 7. rhodesiense of Stephens
and Fantham. The development of the latter has already been described.t+
It will therefore only be necessary here to present the various data in the
form of tables and figures, which can then be compared with similar tables
and figures in the previous paper.
THE DEVELOPMENT OF T. BRUCEI, ZULULAND STRAIN, 1913, IN G. MORSITANS.
Seven experiments were made with laboratory-bred flies. Three of these
were positive and four negative. Two experiments were also made with
wild flies, both of which were positive.
Table I.—Laboratory-bred Flies.
No. of Experiment 2 No. of days Temperature
Date. Hxpt.| flies positive or Ne, Sere. before flies at which flies
used. negative. * | became infective. kept.
1913.
| Feb. 11 | 1857 58 - 1
/ ., 1711909] 50 + 10 21 ?
Mar. 11 | 1988 A5 + 20 13 84° F. (29° C.)
| oy de | LEO 55 — 4,
April 25 | 2111 50 = 3
May 26 | 2188 30 = il
June 23 | 21884; 20 + 8 14 84° F. (29° C.)
* ‘Roy. Soe. Proc.,’ this vol., pp. 493 and 511.
+ Ibid., this vol., p. 516.
Description of a Strain of T. brucei from Zululand. 527
Three hundred and eight flies were used and 47 infected flies were found—
15°3 per cent.
It is difficult or impossible to explain the difference in the ratio of
infected flies. Experiment 1857 has only one infected fly in 58; Experi-
ment 1988, 20 in 45. There is no record as to whether Cage 1857 was kept
in the incubator or not, but it is to be presumed that it was, as was the
habit at that date. From Table III it will be seen that the flies in
Experiment 1988 were fed for eight days on an infected dog, monkey, and
goat. It is possible that this had something to do with the high rate of
infection, but it is impossible to say with certainty. The scarcity of
laboratory-bred flies made it out of the question to pursue this line of
inquiry. Experiment 2188 has also only one infected fly in 30, but this is
capable of explanation. Experiments 2188 and 2188A were carried out for
the sake of economy with the same cage of flies. It having become evident
(see Table IV) that the flies after their first feeding on an infected rat had
failed to infect Monkey 2203, the 20 remaining flies were again fed on an
infected guinea-pig, with the result that eight of them became infected.
Table I1.—Wild Flies.
| |
No.of | Experiment 3 No. of days Temperature
Date. | Expt.| flies positive or ee oF Foscoted before flies at which flies
| used. negative. tes soune | became infective. kept.
1913.
July 22 | 2309) 50 rs 0 34 84°F. (29°C.)
» 26| 2313) 50 es 5 24, 84° F. (29° C.)
In Experiment 2309 none of the flies were dissected, hence no infected
flies were found. In Experiment 2313, only 21 flies out of 50 were
dissected. These experiments are tabulated here as they give the number of
days before the flies became infective, and thus afford data as to the length
of time the cycle of development runs in the fly.
Details of the Five Positive Experiments.
The following table gives the principal details in the carrying out of the
five positive experiments. The first three were carried out with laboratory-
bred flies, the last two with wild flies.
It would appear from these five experiments that an average period of
21 days elapses before the cycle of development of 7. brucei, Zululand, 1913,
is complete in G@. morsitans and the fly becomes infective.
bo
Sm
528 Sir D. Bruce and others. Description of a
Table III.
Expt. Day of expt. Procedure. Remarks.
°
1909 1-9 Flies fed on infected monkey. | Trypanosomes appeared in
10 Starved. blood of Monkey 1970 on
11-29 Fed on clean Monkey 1970. the 28th day. ;
1988 1-8 Flies fed on infected dog,| Trypanosomes appeared in
monkey, and goat. blood of Dog 2007 on the
9 Starved. 20th day.
10-22 Fed on clean Dog 2007. Monkey 2058 showed trypano-
23 Starved. somes on the 30th day.
24-29 Fed on clean Monkey 2058.
21884 1-9 Flies fed on infected guinea- | Trypanosomes appeared in
pig. blood of Dog 2254 on the
10 Starved. 21st day.
11-22 Fed on clean Dog 2254. Monkey 2298 showed trypano-
23 Starved. somes on the 32nd day.
24-27 Fed on clean Monkey 2298.
2309 1-7 Flies fed on infected dog. Trypanosomes appeared in
8 Starved. blood of Monkey 2316 on
9-42 Fed on clean Monkey 2316. the 41st day.
2313 1-4 Flies fed on infected monkey. | 'Trypanoceomes appeared in
5 Starved. blood of Dog 2861 on the
6-15 Fed on clean Monkey 2318. 31st day.
16 Starved. Monkey 2318 never showed
| 17-31 Fed on clean Dog 2361. trypanosomes.
Details of the Four Negative Experiments.
The following table shows the method of procedure in carrying out the four
negative experiments.
In each of them laboratory-bred flies were used :—
Table IV.
Expt. Day of expt. Procedure. Remarks.
1857 1-8 Flies fed on infected monkey. | One infected fly found on the
9 Starved. 62nd day.
10-60 Fed on clean Monkey 1941.
1996 1-9 Flies fed on infected monkey. | Four infected flies found.
10 Starved.
11-55 Fed on clean Monkey 20381.
2111 1-8 Flies fed on infected dog. Three infected flies found.
9 Starved.
10-14 Fed on clean Dog 2100.
15 Starved.
16-80 Fed on clean Monkey 2125.
31 Starved.
32-43 Fed on clean Dog 2189.
2188 1-8 Flies fed on infected rat. One infected fly found on the
9 Starved. 21st day.
10-27 Fed on clean Monkey 2203. .
Strain of T. brucei from Zululand. 529
RESULT OF THE DISSECTION OF THE INFECTED FLIES.
Table V.—Laboratory-bred Flies. Positive Experiments.
Proboscis.
Time, |———_——— | Proventri- Fore- Mid- Hind- | Salivary
(See days. Labial | Hypo- culus. CED, gut. gut. gut. glands.
cavity. | pharynx.
1909 33 - a =
1909 34 - + ++ ++ ++ =
1909 35 = | = ++ ++ ++ ++
1909 36 = ++ ++ ++ ++ +
1909 37 - + + + =
1909 43 - ++ + + =
1909 AT a us oy a
1909 50 me fe pa
1909 51 = = eee fe tt of oth
1909 57 = + =e
1988 22 _ - | aR ae dap || ap ap ++ _-
1988 | 23 a = Ba as f ak
1988 30 = _— + + + —
1988 30 2s s a Fa ae
1988 31 — = ‘A, ve
1988 31 — + + + ar + + ++
1988 31 + - + + + + -
1988 33 es = + x
1988 34 = hs rs a5
_ 1988 34 ft + + fh a
1988 34 _ = + =
1988 34 =_ — + is
1988 34 & a + me
1988 34 = i & a ae
1988 34 = 2 ip re
1988 | 34 a S + a a
1988 | 35 = i +
1988 36 — = + + Bf Bs Kees
1988 37 = — + + + as
1988 | 37 = is sii Ee + + + ue
21884 | 24 we ie + ee
21884 24 — = + i
21884 | 29 a e
21884 | 32 a oe
2188, | 32 is ce
21884 32 + =
21884 32 + + + ++
21884 32 + =
In Experiment 1909, 10 infected flies were found. Three of these had the
salivary glands swarming with trypanosomes ; in none was the labial cavity
or hypopharynx found to contain flagellates.
In Experiment 1988, 20 infected flies were found. Two had an invasion
of the salivary glands. In one it is noted that a few active trypanosomes
were seen in the labial cavity, and in one that a few “blood forms” were
seen in the hypopharynx.
In Experiment 21884, eight infected flies were found, one of which had
530 Sir D. Bruce and others. Description of a
the salivary glands swarming with trypanosomes. Parts of these glands
injected into Rats 2311 and 2312 gave in both cases a positive result.
Table VI.—Laboratory-bred Flies. Negative Experiments.
; |
Proboscis.
‘TM€; || =a | EEL OMe Nbr Fore- Mid- | Hind- | Salivary
ap days. Labial | Hypo- culus. paae. gut. gut. gut. glands.
cavity. | pharynx.
1857 62 _ - ee ee —
1996 43 _ = + + + =
1996 57 _ _ _ ge tb cae rs oper: =
1996 57 | ++ =
2111 12 = = = = = + a =
2111 29 = - | + + ++ ++ ++ —
2111 40 = = ie Pr
2188 | 21 ee i su
| | / |
In none of the negative experiments was an infection of the salivary
glands found. Nine infected flies were dissected, but not one of these had
passed into the infective stage. No parasites were found in the proboscis at
any time.
From a consideration of these tables it will be seen that this strain of
T. brucei, Zululand, 1913, belongs to the same group as 7. gambiense and
the trypanosome causing disease in man in Nyasaland as far as their
development in G. palpalis and G. morsitans is concerned. This develop-
ment takes place in the intestine of the fly and, after a varying number of
days, passes forward into the salivary glands, where the final stage in the
cycle is reached—the infective or “blood forms.” In this group the
parasites are never found fixed in the labial cavity as in the pecorwm and
vivax groups.
THE TYPE OF TRYPANOSOME FOUND IN THE INFECTED FLIES.
Plate 25 represents the developmental forms of 7. brucei, Zululand, 1913,
found in the proventriculus and salivary glands of G. morsitans. A descrip-
tion of the various types found in the different parts of the alimentary tract
is not considered necessary, as they are identical with those found in the
development of 7. gambiense in G. palpalis, which have already been fully
described.*
Figs. 1 and 2 are two long trypanosomes from the proventriculus.
Figs. 3-6 are the same, but they were extruded on to a cover-glass by
* “Roy. Soc. Proc.,’ B, vol. 83 (1911),
Sir David. Brace & others.
£6
Trypanosoma bruce. Lututand 7 DAS.
Development ve Glossina nrorsilans.
VW Bruce. det. X 2000.
Strain of T. brucei from Zululand. 531
a living non-infective fly, and probably came through the labial cavity from
the proventriculus. Some of them may possibly have come from the
hypopharynx, and in that case they may have been proventricular forms on
their way to infect the salivary glands. Figs. 7-20 are various types found
in the salivary glands—crithidial, encysted, and immature “blood forms.”
Figs. 21-28 represent the finished product—the infective or “ blood forms.”
If this plate is compared with that representing the developmental forms
of the trypanosome causing disease in man in Nyasaland* in the proventri-
culus and salivary glands of G. morsitans, the extraordinary likeness between
the two is at once evident, and is another argument in favour of these two
strains being identical.
CONCLUSIONS.
1. 7. brucei, Zululand, 1913, belongs to the same group as 7. gambiense as
regards its cycle of development in the tsetse fly.
2. It has been shown that the trypanosome causing disease in man in
Nyasaland also belongs to the same group.
3. The cycle of development of the Nyasaland and Zululand trypanosomes
in G. morsitans is so marvellously alike that it affords another reason for
believing in the identity of these two trypanosomes.
DESCRIPTION OF PLATE.
Figs. 1 and 2.—Trypanosomes from proventriculus.
Figs. 3-6.—Proventricular types which were extruded on to a cover-glass by a living
non-infective fly.
Figs. 7-20.—Crithidial, apparently encysted, and immature “blood forms” found in the
salivary glands.
Figs. 21-28.—The final stage in the salivary glands—the infective or “blood forms.”
Stained Giemsa. x 2000.
* “Roy. Soc. Proc.,’ this vol, Plate 24.
532
On the Lack of Adaptation in the Tristichacee and Podostemacee.
By J. C. Wiis, M.A., Se.D., etc., Director of the Botanic Gardens,
Rio de Janeiro.
(Communicated by Dr. D. H. Scott, F.R.S. Received February 24,—
Read April 30, 1914.)
CONTENTS.
PAGE
Condutions| of Vile frsc-e-.-ecceseee scence 534
Morphological Structure .................. 536
Geographical Distribution ............... 544
Absence of Adaptation ...................5. 546
Process of Evolution .............--2--ses0s- 548
With the exception of some of the parasitic orders, such as the Bala-
nophoracee, there are probably no families of flowering plants—one might
almost include flowerless—which are so completely transformed from the
average or mesophytic type of the phanerogams into types which are so
completely unique and peculiar, as the Tristichacee and still more the
Podostemacez. Nor are there any in which, with such very great uniformity in
the conditions of life, there is such remarkable variety in the morphological
structure.
The structure of the orders, or rather of their members, being unique, and
the conditions under which they live being also unique, it has been taken for
granted that the former is in a high degree adapted to the latter, the flat
thallus-like expansions of stem or root being looked upon as admirably suited
to the rushing water in which they live. So long as we were almost completely
ignorant of the actual living plants, and content with dead material collected
mainly in the dry seasons, this was all very well, but now that for 17 years I
have devoted much attention to these plants,* have studied them in the living
condition in their natural habitats in India, Ceylon, and Brazil, have followed
them from germination right through their life-history, and in other ways
become absolutely familiar with them, and as a result of all this have
arrived at diametrically opposite conclusions, it will repay us to examine into
the question in some detail.
Evolution is usually supposed to have produced the extraordinary variety
of forms now existing by adapting members of very different families to very
* Willis, “A Revision of the Podostemacez of India and Ceylon,” ‘ Ann. Perad.,’
vol. 1, p. 181 (1902) ; “ Studies in the Morphology and Ecology of the Podostemacez of
Ceylon and India,” loc. cit., p. 267.
Lack of Adaptation mm the Tristichacee and Podostemacee. 533
different conditions of life. The climate, the soil, the competition with other
living beings, many conditions, vary in the most complicated way, and it is
very difficult, if not impossible, to disentangle the effect of the different
factors that may have a hand in producing the result. But in the case of
these two closely allied families we are able to study the problem of evolution
with many of the complication of factors removed or simplified. There is no
competition with other families, for these two have their habitats to themselves.
There is no difference in soils, for all grow on naked waterworn rock. There
is no difference in climate, for all grow in water in the tropical or sub-tropical
zone, and are represented only by seeds in the colder weather. There is no
difference in circumambient medium, for all grow in running water. The
illumination is the same for all. The conditions of their life are the most
absolutely uniform that it is possible to conceive ; even in a laboratory it
would be very difficult to produce conditions more uniform, and they must
have been the same since the original founders of the orders first took to life
in running water.
We cannot for one moment suppose that these plants took to the water at
different stages of evolution into a family upon land; they must obviously
have gone through the whole of their evolution in water from an extremely
early stage, during which the ancestors probably retained a certain power of
surviving upon land. Now the most essential point of this argument is that
here we have two entire families, containing about 30 genera and over 100
species of the most varied morphological structure possible, entirely evolved
under perfectly uniform conditions which cannot have varied, except for all
alike,* since the evolution of the families began. The evolution consequently
cannot have been in any sort of response to a necessity of adaptation to
different conditions, for there are and have been no different conditions to
which to be adapted, since the first members of these families began to live in
running water.
We shall see, first of all, that the conditions of life are absolutely uniform
(2) that the families contain the most astonishing variety of morphological
structure, (3) that all the different stages, so to speak, in the attainment of the
most bizarre of these forms, live together, and that the least bizarre are the
most widespread and common, and in consequence (4) that as there are
no changed conditions to which to be adapted, there can be no adaptation
to conditions other than to the general conditions which are common to
all, and have been common to them since the families began, and (5) that
therefore there can, in all probability, be no selection of infinitesimal variations,
but progress must have been by something of the nature of mutations or fixed
* As, for instance, if the climate became warmer.
534 Dr. J. C. Willis. On the Lack of
changes without natural selection. Lastly we shall consider in brief the
factors which seem to have had an influence in the evolution of these remark-
able families.
The orders Tristichacee and Podostemacez (for I have shown in a recent
paper* that the old family of Podostemacee must be split into these two,
unless some intermediates can be found. in Africa), as is well known,
consist only of annual plants which live upon the surface of smooth water-
worn rocks in rapidly moving water, and are confined, with the exception of
one species found in Ohio, to the warmer regions of the globe. During the
wetter season of the year they carry out their vegetative life, and form their
flowers as the dry weather approaches. The flowers open above the water
level as the rivers sink, and the seeds are shed upon the rocks, where those
few that manage to retain their position germinate with the beginning of the
wetter season.
Conditions of Life—It has long been an axiom that the conditions of life
of the plants of still water are extraordinarily uniform, and that to this is due
the small number of these plants, and the small amount of evolution through
which they appear to have gone and to be going, together with their enor-
mously wide distribution. But it will soon be seen that the conditions of life
for the Podostemacez and Tristichacee are if anything even more uniform,
and yet in contrast to the plants of still water they show the most astounding
morphological differences, and form about 30 genera with over 100 very
different species (and it is certain that very many species and probably genera.
have yet to be discovered). The distribution of these species is more and
more localised the more specialised they are in structure, while the widely
distributed genera are those which are the most like the original ancestors
from which the Podostemaceze and Tristichacez are descended.
The conditions of life of these plants are, and must always have been,
as uniform as, or more uniform than, those of other water plants. In the
first place, as to substratum, they grow as a rule only upon rocks, but, as
they will also grow upon anything which may have become wedged between
the rocks so as to be immovable, the actual chemical or physical com-
position of the substratum cannot be of importance; in all probability the
plants take nothing from it unless, perhaps, silica. They grow upon this
rock bottom by means of creeping roots, which give off secondary shoots.
They are thus absolutely compelled from the start to a plagiotropic
mode of life. In the second place, as to temperature conditions: the
orders are, with the exception of Podostemon ceratophyllwm, found in Ohio,
* Willis, “A New Natural Order of Flowering Plants—Tristichacez,” ‘Linn. Soc.
Journ., Bot.’ (in the press).
Adaptation in the Tristichacee and Podostemacee. 535
confined to the tropics and sub-tropics, and the coldest water in which I
have ever seen any was 57° F. (14° C.), in the Khasia Hills of Assam, the
warmest 81° F. (27° C.). Their temperature conditions are thus more
uniform than those of other water plants. Their conditions of light
supply are also uniform. They live only from the water-level down
to perhaps 50-75 cm. The medium in which they live, the running
water of tropical rivers, is very uniform, and their food supply also, as
it comes from the passing water. The only differences between them
in the numerous localities in which they occur are therefore in the speed of
the water current. The fastest water in which I have found any was in the
Rio Piabanha, north of Petropolis, near to Rio de Janeiro, where Lophogyne
arculifera was growing in water going from 24 to 4 miles an hour, and the
slowest was at Hakinda, near Peradeniya, in Ceylon, where Farmeria
metzgertoides and Podostemon subulatus were growing im an eddy that moved
perhaps half a mile in the hour. But, if this involved any difference in
structure, there would surely be more “ Zugfestigkeit ” in the thalli of the
more rapid water, and an examination of the various sections given in my
monograph of the-Indian forms will show that this is not so—some have it,
some not.
It is true that at any one actual spot, of, say, a square yard, on a rock,
there are most commonly only one or two species to be found growing, but
this does not mean that no others could grow there, but simply that each
one has some slight preference in positions. Always there is more or less of
intermixture. For instance, from my Indian monograph I may quote the
various species, with those which were recorded as found together with them :—
Tristicha ramosissima : Griffithella Hookeriana.
Lawia zeylanica : Hydrobryum olivaceum, lichenoides, Furmeria metzgerioides,
Podostemon subulatus, Dicrea stylosa, Grifithella Hookeriana.
Podostemon subulatus: Dicrea elongata, stylosa, Farmeria metzgerioides,
Lawia zylanica, Hydrobryum olivacewm.
Podostemon Barberi: Griffithella Hookeriana.
Dicrea elongata: Podostemon subulatus, Dicrea stylosa, Hydrobryum
olivaceum.
Dicrea dichotoma : Hydrobryum olivaceum.
Dicrea Wallichii: Hydrobryum lichenordes.
Dicrea stylosa: D. elongata, Podostemon subulatus, Hydrobryum olavaceum,
lichenoides, Lawia zeylanica, Griffithella Hookeriana.
Grifithella Hookeriana: Lawia zeylanica, Podostemon Barberi, Dicrea
stylosa, Tristicha ramosissima.
Willisia selaginoides : Hydrobrywm lichenoides.
536 Dr. J. C. Willis. On the Lack of
Hydrobryum lichenoides: H. olivaceum, Willisia selaginoides, Lawia
zeylanica, Dicrea Wallichir.
HZ, sessile: ?
A. olivaceum: Lawia zeylanica, Farmeria metzgerioides, Dicrea stylosa
elongata, dichotoma, Hydrobryum lichenoides.
Ai. Griffithii : 2
Farmeria metzgerioides: Lawia zeylanica, Hydrobryum olivacewm, Podo-
stemon subulatus, etc.
Farmeria indica : ?
When we remember that often a certain species does not grow in the
same district as another species, and that these are only a few observations,
it is pretty evident that almost any species could live with almost any other,
or in almost any place. In Brazil I have found the most incongruous-
looking species, such as Zvisticha hypnoides and Apinagia Riedelir, or
Mniopsis Weddelliana and Mourera aspera, growing side by side, inter-
mixed, in two or three localities. Further, in the State of Rio, their habitat
is nearly always shared to some extent with a moss, which appears to be
able to survive on the dry rock if exposed to the air by the fall of the water.
As regards other conditions of life, it may be pointed out that these
plants escape to a large extent from competition with other plants, for,
except for an occasional moss or fern, nothing else is ever found on the
rocks with them, and, owing to the enormous destruction of seed before
germination, there is little competition among themselves. They are to
some considerable extent attacked by such animals as can get at them, for
they are very rich in starch towards the end of the season.
Their conditions of life, then, are in the very highest degree uniform, but
in no sense can this be said of their morphological construction, which is the
most various and complex that one can conceive. By no stretch of imagina-
tion can the variety in the conditions of life be made to fit one quarter of the
variety of structure. While the conditions of life and the variations in
those conditions are the same in Brazil, India, and Africa, and certain
almost identical species occur in all these places, the general trend of the
morphological construction in India is towards flattened primary roots, in
Brazil towards flattened secondary shoots, and in Africa, so far as our ab
present extremely limited knowledge goes, sometimes, at any rate, towards
flattened roots combined with tall stems.
Morphological Structwre-—Before going further, as these plants are not very
familiar to most botanists, it will be well to sum up in brief some of their
morphological peculiarities. Taking first the family Tristichacez (Tristicha,
Lawia, and Weddellina), be it noted first of all that the members of this
Adaptation in the Tristichacee and Podostemacee. 537
family, though only, many of them, slightly modified from “common ” types,
occupy the same positions as the highly modified members of the Podostemacez
proper, whether in swiftly moving or in slowly moving water. Now if such
simple forms as these suffice to occupy the positions, there can be no adapta-
tional need for the complex forms of the Podostemacez proper. But to return
to the structure: the most “primitive” -looking species is undoubtedly
Tristicha ramosissima Willis, which grows in South-West India. The primary
axis of this plant is not known, but as the creeping roots are just like those
of other forms, it may be taken for granted that it gives rise to creeping
closely attached roots, running over the rocks in all directions. Upon these
are borne endogenous “secondary ” shoots, which in this plant grow out in a
fairly normal way to a length of a foot or so. They are not dorsiventral, and
bear the flowers, which also are not dorsiventral, but have the formula
P3, A3, G(3), and are perfectly regular. A feature of the plant is the numerous
“yamuli” or shoots of limited growth, which here are in no sense dorsi-
ventrally arranged or constructed. Now in this plant the only adaptations,
if adaptations they can be called, to the mode of life, are (1) the great and
regular development of secondary shoots on creeping roots, and (2) of haptera
or organs for clinging to the rock, and (3) the absence of intercellular spaces.
But I have no hesitation in affirming that this plant could live quite com-
fortably in any of the localities occupied by the members of the two families,
with the exception of those in which during the growing season the water was
liable to be less than an inch or an inch and a half in depth. Life in
shallower water is quite easy for another species of Tristicha—hypnoides.
But in this one begins to see increasing dorsiventrality in the plant. Once
placed upon a rock, in which downward growth is impossible, and dorsi-
ventral or plagiotropic direction of growth ensured by the creeping roots,
the impetus to dorsiventrality appears to be given, and the whole further
evolution of both families seems to be chiefly in the direction of increasing
dorsiventrality, both in the vegetative and the floral organs. In 7. hypnoides
the main secondary shoot begins to show some dorsiventrality and the ramuli
have become very distinctly tristichous, with one upper and two more lateral
rows of leaves. The flower is also dorsiventral in having lost the upper and
one lower stamen. In a new species of Tristicha which has lately been
discovered near to Rio by Snr. F. Tamandaré de Toledo, the ramuli are
not only tristichous, but lie down flat upon the rock, and have the upper
row of leaves smaller than the laterals, while the secondary shoots have
become flat expanded “stem-thalli.” Leaves are borne on the upper
surface of the flat expansion (which les flat upon, and is attached to, the
rock), and ramuli as well as flowers on the margins. The flowers have
538 Dr. J. C. Willis. On the Lack of
only the two lower stamens. Now this species can live quite happily in
5-8 mm. of water, and again I may repeat that these three Tristichas
could perfectly well occupy all the localities of the Tristichacee and
Podostemaceze, the conditions being identical throughout. In actual fact
they, with the other species of the genus—Z. alternifolia—do oceur in a
- very great proportion of the places in which plants of these families have
been found, throughout the distribution zone of the genus Tristicha, which
is almost coterminous with that of the orders.
Weddellina, a genus confined to the north of South America, has a
structure very similar to that of Z. ramosissima, and a symmetrical
flower.
Lawia, the remaining genus of this family, departs, so to speak, upon a side
line which was indicated by the secondary shoots of Z7’risticha sp. nov., but
here it is the primary axis which flattens out into a broad prostrate thallus,
which does not bear ramuli like the rest of the order and has symmetrical
flowers like 7. ramosissima. The genus is confined to the region from Ceylon
to Bombay, and has only one species, L. zeylanica, which occurs in many
forms, differing slightly, so far as can be observed, in every locality.
Before going on to deal with the Podostemacez proper, it may be well to
repeat that the Tristichacez can, and do, live quite happily in every locality
occupied by the other family, and that the genus Tristicha, the typical genus
of the family, and the least modified (in one at least of its species), is the
most widely spread of all the genera of both families. The whole family
Podostemaceee, with its remarkable morphological constructions, is therefore
adaptationally unnecessary. All its extraordinary features are de lume, and
cannot have arisen in response to any need for adaptation to different
conditions, for there are no different conditions to which to be adapted,
and none of the highly modified forms is so successful, or so common, as
T. hypnoides, which has only a very slight modification from the original type
from which the family is descended. We cannot suppose that all these
changes had to go on just to fit the family to live in running water, or
the first members of it would never have been able to survive, and the
Podostemons, etc., which are only slightly modified, would not be the most
widespread and common of all to-day.
To pass on now to the Podostemacez proper (tribe Achlamydatz of the
older writers), we may run very briefly over the striking morphological
differences to be found in the family. They are mainly of two kinds—
flattening of the secondary shoots and flattening of the primary roots. The
least modified types In many respects are the South American forms Ligea
{(fnone), Marathrum, and Mourera. All show, however, rather more dorsi-
Adaptation in the Tristichacee and Podostemacee. 539
ventrality than do half of the Tristichacee. They have creeping roots which
give rise to secondary dorsiventral shoots with large leaves developed in two
ranks, The flowers are radially symmetrical, but are liable at times to become
slightly dorsiventral by the abortion of some of the stamens. The inflorescence
in Mourera is flat.
A further stage in this direction is shown in the South American
Lophogynes and Apinagias, where the secondary shoots, while smaller in
size, have become more or less flattened out and thalloid by the union of the
bases of the leaves or the flattening of the stem. The flowers are usually
markedly dorsiventral by the disappearance of the upper stamens. An
extreme case of this construction is afforded by Castelnavia, where the
| (secondary) thalloid shoots lie flat upon the rock, like the (primary) shoots of
Lawia in the other order.
Another section of the family is represented by the Podostemons,
occurring in Asia and America, and probably in Africa also, where the root
is more or less cylindrical and creeping, and gives rise to secondary shoots
which are only slightly dorsiventral, chiefly in their branching, and which
bear highly dorsiventral flowers. Allied to this genus, as well as perhaps to
Lophogyne, etc., is Mniopsis, which is confined to South America, has
a thallus like Lophogyne, and a flower like Podostemon.
Spherothylaxz abyssinica Warming (Abyssinia) has what is apparently
a primary axis that grows up to a considerable height, bearing compound
leaves and flowers, while at the same time the root at the base forms a flat
thallus, something like a lichen or a liverwort, bearing secondary shoots
which remain very short, and ultimately become floriferous also. The
flowers are highly dorsiventral. In A. olivacewm and other species of
Hydrobryum (India and Ceylon) the primary axis is much reduced, only
seldom bearing flowers, but the flat lichen-like thallus or root is large, and
gives rise to great numbers of secondary shoots, which ultimately become
floriferous, with very markedly dorsiventral flowers.
In Dicrea and Griffithella the root flattens out, sometimes in long thread-
or ribbon-like forms, sometimes in more condensed shape, sometimes even
forming a goblet-like organ. On the edges are borne the secondary shoots,
which are very short, lengthening slightly at the end of the season, when
they bear each a dorsiventral flower.
In Farmeria (India, Ceylon) the roots are creeping and flattened, as in
Hydrobryum, but the flower carries the dorsiventrality to the last stage,
showing it in the interior of the ovary, and in the embryo. In one species,
Ff. metzgerioides Willis, the fruit contains only two seeds, and does not
dehisce, the seeds germinating im situ on the rock. This is the only species
540 Dr. J. C. Willis. On the Lack of
in either family which has a proper arrangement for preventing its seeds
from washing away.
In none of the species with flattened highly dorsiventral root does the
stem or shoot of the secondary shoots show any sign of flattening out, while
in none of those with flattened secondary shoot does the root show sign
of flattening. The former are mainly characteristic of India and Ceylon, the
latter of South America.
For the great variety in detail which all these plants exhibit reference
must be made to the work of Warming* and to my previous work (Joe. cit.)
published upon the Indian and Ceylon forms. Tulasne’s monographt also
contains a fine set of pictures of the families.
We may now proceed to deal in more detail with the various structural
features of these families, which have generally been looked upon as adapta-
tions. In the first place, let us take the flattened thallus-like roots or
secondary shoots. These have been generally regarded as adaptations to
withstand, or to avoid, the rush of the water. But, as already pointed out,
the forms that are exposed to it have no marked “ Zugfestigkeit.” In my
monograph of the Indian forms I expressed the opinion that the adaptation
was rather to shallow water than to rushing water. The most highly
adapted form, on either view, is H. olivacewm, and yet this very form,
at the period when the rush of the water is most violent, possesses a tall
primary axis with a great bunch of leaves at the end, an axis, moreover,
which can hold fast if the flat creeping thallus be removed. Until I came
to Brazil I was still in some doubt as to whether these dwarf forms and
closely attached thalli might not be regarded as in some degree at any rate
adaptations to rushing water, but what I have seen here has completely
destroyed that idea, and enabled me to write this paper. Here there are no
flat creeping root thalli, but instead there are, as in M. Weddelliana,
flattened secondary shoots, forming thalloid outgrowths. Now these thalli,
though with far less holdfast than the Indian root thalli, live in more rapid
water. Never or very rarely, though for many years I have been familiar
with the habitats of the eastern forms, have I seen any in such rapid or violent
water as the forms which grow in the State of Rio de Janeiro. I have even
found Lophogyne arculifera with the water falling on to it from a measurable
height. Even the large Brazilian species of Mourera, Apinagia, and
Marathrum live in very swift water without any difficulty, as may be very
strikingly seen in Plate 62 of v. Wettstein’s ‘Vegetationsbilder aus
* Warming, “ Familien Podostemacee, J-VI,” ‘Kgl. Dansk. Vidensk. Selsk. Skr.,’
6 raekke, ii, 1881; ii, 1882; iv, 1888; vii, 1891; ix, 1899; xi, 1901.
+ Tulasne, ‘Monographia Podostemacearum,” ‘Arch. du Mus. d’Hist. Nat.,’ vol. 6 (1852).
Adaptation in the Tristichacee and Podostemacew. 541
Stidbrasilien.’ The conditions of life are so absolutely uniform that any
species can live in practically any place affected by these orders, though at
any one locality they will more or less group themselves each in the kind of
place that best suits it.
It therefore appears to me in the highest degree improbable that either
primary root or secondary shoot thallus can be looked upon as an adaptation
-to violent water, especially when we consider that one form is mainly
Asiatic, the other mainly American, while, further, they both share their
habitats with the very slightly modified Tristichas, and with the also slightly
modified Podostemons. As I have before stated, the adaptation, if adapta-
tion there be, is to shallow, not rushing, water, but even upon that it is as
well not to lay too much stress, because 7’. hypnoides and some species of
Podostemon, neither of them with any thalloid flattening, are as well suited
to, and grow in, the same shallow water as any of the thalloid forms.
The only thing that it is safe to say, after 40 years during which opinion
has at first gone headlong in favour of adaptation and afterwards against it,
is that the small forms can live in any depth of water in which the condi-
tions of illumination will allow their life to go on, while the very large
forms are only to be found in the larger rivers.
If all the extraordinary morphological differences between these plants
were to be regarded as adaptational to the extremely small or non-existent
differences in their conditions of life, the adaptation in land families living
under more variable conditions would have to be something positively
astounding. Or again if these differences are adaptational, why do we get
one kind in one country, another in another, though there are similar species
of the same genus living in both? Or why in one genus (Hydrobryum)
do we get “root” thallus, in another (Lawia), living in the same place in the
same river, “shoot ” thallus ?
To pass on now to the flower. In the Tristichacee, which are the
less modified forms, it is simple and usually regular, some species of
Tristicha itself having only one or two stamens on the lower side. In
the Podostemacee it is sometimes regular, but more often dorsiventral,
frequently to a very high degree. Now dorsiventrality has often been
supposed to be an adaptation to insect visits; it is usually supposed only to
be found in lateral flowers and in flowers which stand horizontally when
open. But in the Podostemacez it is at its highest degree, and that the
most extreme known in the higher plants, in flowers which are anemophilous,
terminal, and erect. In my monograph of the Indian forms I have gone
fully into this question, and may refer to that work for details. The con-
clusion there reached was that “the dorsiventrality of the flowers, which is
VOL. LXXXVIIL—B. Zn
542 Dr. J. ©. Willis. On the Lack of
the most important morphological character in the classification of the
order, is a direct result of, or in direct correlation with, that of the vegeta-
tive organs, being greater the greater the dorsiventrality of the latter.”
And again “it seems then not unreasonable to conclude that the dorsi-
ventrality of the floral organs has been, so to speak, foreed upon them by
that of the vegetative organs or by their position upon the latter without
any reference to advantages or disadvantages to be derived from it in the -
performance of the functions of the floral organs themselves. The only
demand made upon them, so to speak, has been that they should not cease
to set seed.”
A comparison of the characters used for separating the genera of
_ Podostemacez proper, ¢.g. by Warming in Engler and Prantl’s ‘ Natiirlichen
Pflanzenfamilien, shows at once that practically all these characters, ¢..,
suppression of the stamens of the upper side of the flower, obliquity of the
gynceceum, mode of opening of the spathe, etc., are simply expressions of,
and involved in, the degree of dorsiventrality of the flower. As I regard
this, as above reiterated, as being due to a large extent, at any rate, to the _
vegetative dorsiventrality, I have consequently employed vegetative characters
in addition to the floral in determining the Indian genera, and in the work
which I am carrying out upon the South American forms I propose to do
the same.
By ‘no stretch of imagination can the dorsiventrality of the flowers be
regarded as adaptational, and I have elsewhere shown good reason to suppose
that in general it follows that of the vegetative organs. But in this paper
we have seen reason to believe that that of the vegetative organs also is
not adaptational. No gain whatever comes to these plants, in other words,
from all their wonderful morphological changes, and the whole differentiation
of the two orders into nearly 30 genera with numerous species is almost
entirely an expression of the dorsiventrality forced upon them by their
plagiotropic growth. The least dorsiventral and least modified species of
the least modified genus of the Tristichacee can and do live in nearly all the
places occupied by the two families, and the whole differentiation of the
families is merely de luxe, and without any adaptational signification what-
ever.
Finally, let us consider the seed. In most of the plants of these orders
there are very numerous (200-600) minute seeds, whose outer coat becomes
mucilaginous when wetted, but in some the number is much reduced,
especially in Farmeria, where in one species there is a dehiscent fruit with
about four seeds, in the other, /. metzgerioides Willis, an indehiscent fruit
with only two, which germinate 7m situ.
Adaptation in the Tristichacee and Podostemacee, 543
Now if these plants were really adapted to their habitat, one of the first
things one would expect to find in them would be some arrangement to
enable the seeds to cling to the rocks upon which they find themselves shed.
But there is only one species in which this is the case, F. metzgerioides,
mentioned above. All the rest have capsules which open in dry air and
shed the- seeds upon the rocks. When a shower of rain comes the
mucilaginous layer of the seed swells, and as it dries it attaches the seed
firmly to the rock. This has been looked upon as an adaptation for clinging
to the rock by many people who have forgotten that when again wetted,
as for instance by the rise of the water in the rainy season, the mucilage
again softens and the seed washes away. The seeds once afloat have but a
small chance of arriving at suitable growing places, for if dropped in quiet
water they will not grow, and if carried to rocky places it is very unlikely
that they will catch in anything to form a place for germination. Seedlings
I found in Ceylon to be extremely rare, excepting only in /. metzgerioides.
Only when a seed gets caught in a crack in a rock, or in a hole in the old
thallus, does it get any chance to grow. As soon as it germinates it produces
root hairs to fasten it to the substratum, but even these are often not pro-
duced quickly enough, and I have found, in trying to germinate seeds in
the Botanic Gardens in Rio de Janeiro, that even when the cotyledons are
open, and a number of root hairs attached to the rock, they may yet be
washed away. My experience as yet with germination trials leads me to
suppose that it takes at least from 500 to 1000 or more seeds to give three
or four seedlings, and of these, perhaps, one may come to maturity. This
being the case it is impossible to say that the order—except in the one
species F. metzgerioides—shows any adaptation in this, one of the most
essential things, one would think, in which it might, with its wealth of
variety in structure, be adapted. Even in F. metzgerioides it is by no means
certain that the indehiscent fruit is an adaptation. This form is the most
highly dorsiventral of the whole family, and the peculiar feature of the
indehiscent fruit may be merely another expression of the increasing dorsi-
ventrality which runs throughout the Podostemacee.
After a consideration of all the features of these orders, then, we come to
the conclusion that any and all of the adaptations that there may be in-them
are to be found in 7’. ramosissima, the least modified of all. As we pointed
out at the commencement of the paper, in this species (and the same features
appear throughout the two families) these features are—(1) a great and regular
development of secondary shoots upon creeping, closely attached roots;
(2) the development of haptera or clinging organs to attach the plants to
the rock; and (3) the absence of intercellular spaces. Beyond these there
27 2
544 Dr. J. C. Willis. On the Lack of
are practically no characters in the orders that can possibly be regarded as
adaptational, and most certainly not the characters—of thallus formations,
dorsiventrality of flower, etc.—which really characterise the orders, and
enable them to be separated in the natural system. But are even these
characters which we have enumerated really adaptations? The development
of secondary shoots upon roots is by no means uncommon, though not usually
carried to such a pitch of regularity and perfection as here. The absence of
large intercellular spaces may be directly ancestral; if the orders came, as is
quite possible, directly from plants growing upon the banks of the water,
their members may never have had large intercellular spaces at all, these
being quite useless in their mode of life. Haptera, perhaps, are really
_ adaptations, as they are all but unique, and appear to be modified root
branches, but they form the only feature in the two orders that one can point
to that is at all probably an adaptation. |
Geographical Distribution—Another argument in favour of our main con-
tention may be derived from the geographical distribution of the Tristichaceze
and Podostemacee. The genera which are widely distributed are Tristicha*
in the one family and Podostemonft in the other, both of them genera which
are comparatively little modified from the earlier types of the orders. Of the
other genera the distribution is, roughly, the following :—
“UAW ac eseccee cece cocten W. India and Ceylon.
Weddellina oo... oss... 00 Guiana and N. Brazil.
En One. ake cance gnesdae Guiana and N. Brazil.
Manathrumiincscce sees: Mexico to Brazil.
iRhyncholacisipeesecs- eee Guiana.
AMIMNGCTA .tactiecceee copes Guiana and Brazil.
ligphooyne Vir. aes Province of Rio de Janeiro.
Mourenaitcas conscteeeencee Guiana, Brazil.
Lonchostephus ......... River Amazon.
TaCiSis, 2 einccheconsimencnee River Amazon.
Dicreea sac nosscrasenieee sce Madagascar, India, Ceylon.
iydrobryumn ere: sees India, Ceylon.
Ceratolacis ........ SRE Brazil.
Mmiopsisenrences essen Brazil.
Oseryais aeiaanuntae te Brazil to Mexico.
Castelnavia ..........+..+. Brazil.
* Mexico, S. America, Africa, Madagascar, India.
+ Ohio to Argentina, Ceylon, India, probably Africa.
Adaptation in the Tristichacee and Podostemacee. 545
Cladopusimmniseenn etna Java.
Wallistatin, \e5tsik eee abe W. India, Burma.
Grifiithella vast eer W. India.
Barmera, seeeee..ae. S. India, Ceylon.
The African species are as yet too little known to make it possible to give
their distribution; the genera to which they really belong have yet to be
made out in many cases, but are almost certainly all, or nearly all, different
from the American and Indian.
In other words, the only widespread genera are the non-specialised ones,
while the more specialised the genus, on the whole, the less is its area of
distribution. The non-specialised forms live everywhere with the specialised,
and are every whit as well suited to the positions, which show no differences
in general conditions of life. This result may well be compared with that
which I traced for the Dilleniacez* in a previous paper, being exactly parallel
with that.
Or, again, take the well-known fact that in the Podostemacez the species
are usually very local in distribution, as has frequently been pointed out.t+
The best known instance is Castelnavia, where in the same river, the
Araguaya, some of the species differ at every cataract. Now, in the case of
different species of a genus on land, it has been customary to say that they
have been evolved to suit different mixtures of the conditions of life, and
the absence of many species of one genus in most plants of ‘still water
has been put down to their uniformity of conditions. But here this explana-
tion will not hold. The physical conditions of life at all cataracts in the
Araguaya are the same, and there is no mixture with other forms of life at
all. And this must have been true since the foundation of the family. And
yet the Araguaya contains seven species of this one genus, a genus, moreover,
which is almost confined to this river, in which there occur besides only one
or two species of Oserya and Apinagia. It is another expression of the fact
to which attention has often been called,{ that isolation, as isolation,
favours the production of new species. Why this should be so, we cannot at
present say, but the fact remains. These species of the Araguaya are, of
course, each endemic to its own few waterfalls, and to them may be applied
* Willis, “The Geographical Distribution of the Dilleniacez as illustrating the Treat-
ment of this Subject on the Theory of Mutation,” ‘Ann. Perad.,’ vol. 4, p. 69 (1907).
+ Weddell, “Sur les Podostemacées en général et leur Distribution Geographique en
particulier,” ‘ Bull. Soc. Bot. France,’ vol. 19, p. 50 (1873) ; Goebel, ‘ Pflanzenbiologischen
Schilderungen,’ vol. 2, pp. 331, 374; Willis, ‘Studies in the Morphology and Ecology of
the Podostemaceze of Ceylon and India,” ‘ Ann. Perad.,’ vol. 1, p. 450 (1902).
t Willis, “The Floras of Hilltops in Ceylon,” ‘Ann. Perad.,’ vol. 4, p. 135 (1908).
546 Dr. J. C. Willis. On the Lack of
the remark which I have already made* about the endemic species of Ceylon,
that (p. 13) “in general they have characters which are, so far as one can
conceive, useless in the struggle for existence; they occur in places where
that struggle cannot have been very keen, or between very large numbers ;
they often occur alongside of their most nearly allied species, and very often
the differences in character are such as can hardly conceivably have arisen
by the selection of infinitesimal variations.” The whole argument of that
paper should be read in connection with this, as it produces evidence
to the same end from a study of quite a different nature from that here
dealt with.
Absence of Adaptation.—By a consideration of all the facts which have been
brought forward, we are thus forced to the conclusion that as there are not,
‘and have never been, any changed conditions to which these families have or
had to be adapted, there cannot have been in them any adaptation to changed
conditions after the first adaptation which enabled them to live on the rocks in
running water. The whole of the extraordinary morphological changes
through which they have gone are without any adaptational significance
whatever. The conditions of life under which TZ. ramosissima exists are
the same as those under which the rest of the families exist, and have
always existed since the evolution of the families began. In spite of the great
variety of form and structure, it is impossible to say that any one form is better
suited to the conditions of life than any other. The most “primitive ” forms,
Tristicha and Podostemon, which have neither flattened shoot-thallus nor
root-thallus (except in one species of Podostemon, which perhaps should form
another genus) are the most widespread and the most common.
It may be objected, perhaps, that the modification and adaptation to the
simple change of conditions involved by getting into the water is still going
on, and is enough to account for all the modifications that the families have
undergone. However well A may be adapied, it is of course obvious that B
may be better adapted. But if it requires the evolution of two whole families
with 30 genera and over 100 (perhaps over 200) species of the most various
form to meet this need, we have a very remarkable case before us, when we
consider that the first founders of the families must have been adapted to life
in running water, or the families could never have commenced. If this were
to be the case, we should expect enormously greater variety than actually
exists among the other plants of the vegetable kingdom.
As has been pointed out, the most obvious feature in these plants where, if
* Willis, “Some Evidence against the Theory of the Origin of Species by Natural
Selection of Infinitesimal Variations, and in Favour of Origin by Mutation,” ‘ Ann.
Perad.,’ vol. 4, p. 1 (1907).
Adaptation in the Tristichacee and Podostemacee. 547
anywhere, we should expect adaptation, viz. the attachment of the seeds to
the rocks, shows none, except in one species only, and that the most highly
modified species in the whole family. Unless the haptera be an adaptation,
this is the only “adaptation” in the whole group, and if it takes all this
amount of evolution to get to one adaptation, there cannot be much selection
of advantageous variations. The morphological changes in these plants are so
large and striking that if there were any adaptation involved in them, there
would surely be some evidence to prove it, but there is none, and in all my
work on these plants I have not knowingly suppressed any evidence one way
or the other, or failed to put down anything that I have observed.
Not only so, but the forms that are most like the common ancestors, and
possess none of the thalloid structures of the more bizarre genera, viz.,
Tristicha and Podostemon, are much the most widespread and the most
common. As with the Dilleniacez and other orders instanced in my previous
papers, it is much simpler to regard these as parent genera which have split
off the others by mutation in different places. Everywhere they live together
with the more complex and modified forms, and in equal abundance. None of
the latter have one quarter of their range.
Again, the more complex forms have flowers which are modified in a
direction that can only be looked upon as disadvantageous, ifanything. Ihave
already gone into this question in another paper, and need only refer to it
here. By no conceivable advantages could the flowers have been the subject
of natural selection.
There is no adaptational need for the complex forms; if they are better
suited to the general conditions of life of the orders, there is nothing to prove
it, and they are not a conspicuous success, except in places that happen to
prove exactly right for them, as for instance Lawia zeylanica in the shallow
streams of the Bombay Ghats. The evidence of the local endemic forms is
also against this view of still-progressing adaptation.
The conditions of life, as has been pointed out, are as nearly as possible
absolutely uniform, much more so than in other water plants, which have a
certain amount of soil differences, competition with plants of other families,
and so on, to face. So great is the destruction of seed before germination in
these families that until quite late in the season there is usually ample space
upon the rocks, so that there can be but little competition between them for
space. Competition for food cannot exist in water running so rapidly.
The only “adaptation” that can be conceived of as going on in these plants
under these circumstances is to the ever-present force of plagiotropism, but if
this be admitted, it is also admitted that there can be definite evolution-
factors, which it is one of the objects of this paper to demonstrate. Adaptation
548 Dr. J. C. Willis. On the Lack of
by natural selection to this force, with the enormous destruction of seed that
goes on, is almost inconceivable, however.
The Process of Hvolution—It follows.from the above that, as there is no
adaptational selection, or selection of characters better suited to the struggle
for existence, the process of evolution in these families from the simpler to
the more complex forms cannot have been by the gradual accumulation of
infinitesimal variations, unless these can also be selected by the one
permanent cause acting upon the families, viz., their plagiotropism. Many
of their variations, too, are not dorsiventral, and plagiotropic life would
not be likely to select them. The more complex and modified forms
are in no way whatever superior to the simpler forms; both live together,
_ and one shows no sign of exterminating the other. It is all but impossible,
however, to imagine that plagiotropic life could select infinitesimal variations,
and as I have shown in dealing with the endemics of Ceylon that mutation
is the only possible explanation of them, so here I propose that we accept
the theory of mutation as the only feasible explanation at present possible of
the facts with which we have to deal. Mutations not being liable to go back,
it is possible to accumulate them, unless any one of them should prove of very
serious disadvantage, in which case it would be eliminated by natural selection.
Taking the evidence which is here brought forward with that which I
have set forth in other papers already quoted, added to the fundamental
work of de Vries, it seems to me that a good case is now fully made out for
mutation, and that the onus of proof is completely thrown upon the
infinitesimal variationists, while a very fair case is made out for mutation
without natural selection. Analogy of the conditions and phenomena which
we have been considering in this paper with those which occur throughout
the vegetable kingdom lead almost irresistibly to the conclusion that
mutation without natural selection must be a theory of general applicability,
although there is no reason to exclude natural selection from operation upon
a smaller scale than that for which it has hitherto received credit—chiefly,
be it remarked, it operates by destroying disadvantageous variations, unless
they are compensated by advantageous ones at the same time. This subject,
however, leads into great issues, which must be left for subsequent con-
sideration. A réswmé of the arguments which have led me to take up the
position I hold in regard to this question is given on p. 208* of the third
edition of my ‘Manual and Dictionary of the Flowering Plants and Ferns,
1908.’
The Mechanism of the Process of Evolution—So far the reasoning has been
straightforward, and we have come to the conclusion that there must have
been evolution by means of mutations, and without natural selection in the
Adaptation in the Tristichacee and Podostemacee. 549
ordinary meaning of the term. When we come to consider what this really
means, we are met by great difficulties.
There is one, and apparently only one, or at most two, permanent
deflecting factors, that act now, and have always acted since the evolution of
the families began, when their first ancestors took to life in running water.
These are, first and foremost, their plagiotropic method of growth, forced
upon them by the fact that they live only upon an unyielding substratum ;
they have not, and can never have had, primary roots going downwards into
the rock, and are thus, one might almost say, cut in half, or deprived of
one-half of their polarity. The other factor is the longitudinal strain of the
water, which to a large extent acts in the same general direction.
Now there is also one, and apparently only one, or at most two, permanent
results visible in the evolution of the members of these families, viz., the
plagiotropic or dorsiventral habit both of the growing organs and the
structure of the flower, which is by far the most striking feature in them ;
added to this is the considerable elongation and division of the leaves (or
root-thalli) in some of the forms. Their differentiation into genera and
species depends to a large extent upon these two features, which are merely
expressions of increasing dorsiventrality, and increasing size and division of
leaves (or root-thalli).
Now, when one sees in a family where the complexity of the ordinary
evolution process is so much simplified as we have seen it to be here, one
principal and one subsidiary deflecting factor, and one principal and one
subsidiary result, it is very difficult to avoid the conclusion that these are
cause and effect. But it must be again insisted upon, and clearly understood,
that there has been no selection of advantageous variations. All the forms,
whether slightly or highly dorsiventral, can live in practically all the
localities affected by the families, so that the increased dorsiventrality of
the more modified forms is of no advantage to its possessors. The least
modified forms are the most common and widespread (and this by the way
appears to be the general rule in other families, as I have already pointed
out), while the much modified ones are local.
This being so, we are almost forced to believe that a definite deflecting
factor can make evolution go more or less in a definite direction, without
regard to any advantages to be gained by it, though any modification that
was seriously disadvantageous would be weeded out by natural selection.
But now comes a serious difficulty. If we have to admit that
evolution can take place without natural selection, and that there is no
selection of advantageous variations, why do we (so far as present evidence
goes) not get growing together all the stages in the evolution of the very
550 Lack of Adaptation in the Tristichacee and Podostemacee.
numerous species of these families? And why are there any gaps between
the species? Why are they not filled up by intermediate forms, only differing
from one another in yery slight degree? (If gaps once arise between species,
larger ones will inevitably, so far as one can see, arise between genera, so that
we may leave the genera out of account.) Does this mean that mutations
may be larger than we usually imagine? Or does it mean that a mutation
in one direction involves further mutation in the same direction? Or what °
does it mean ?
It would scarcely seem, perhaps, as if the actual direction of mutation could
have been selected by the forces acting on these plants, unless in the
progenitors there appeared several mutations in different directions involving
others in the same directions, all of which were selected.
As one can almost say that no variations other than dorsiventral ones or
variations in the direction of longer or more divided leaves or thalli, have been
perpetuated, and as the dorsiventrality is no advantage, and as there is no
natural selection except in the direction of extermination, it is very difficult
to escape the conclusion that the evolution was more or less guided in a
definite direction by the plagiotropism. Perhaps this force or natural
selection exterminated variations, other than slight, in other directions.
On the whole, we are inclined to think, though as yet with great diffidence,
and with an open mind, that the evolution of these families was by indis-
criminate mutation, or mutation in every direction, without natural selection,
the mutations in the direction of dorsiventrality and perhaps in the direction
of longer and more divided leaves being on the whole more easily perpetuated
than others—many of which would be killed out by natural selection—and
this on account of the permanent deflecting forces acting on these plants,
and which we may perhaps call their evolution-factors. A strongly marked
evolution-factor, like plagiotropism in these families, can compel evolution to
move on the whole in a definite direction, without any reference to the
advantages or disadvantages to be derived from so moving.
To accept this result as general will explain without any difficulty the
presence of such countless numbers of useless characters in plants,* and may
help to account for the great changes in the botanical landscape which seem
often to accompany the greater changes in the geological landscape—the
great change of conditions on getting into plagiotropic water life seems
to have produced the great variety in the plants we have been considering,
and perhaps the greater landscape changes were similarly accompanied by
great changes of conditions.
* Of. De Vries, ‘The Mutation Theory,’ vol. 1, p. 208 (Delbceuf), 1912.
551
The Action of Certain Drugs on the Isolated Human Uterus.
By James A. GuNN.
(Communicated by Prof. C. S. Sherrington, F.R.S. Received March 7,—Read
April 30, 1914.)
(From the Pharmacological Laboratory, Oxford.)
I have elsewhere shown* that, in experiments on the isolated mammalian
heart, it is perfectly possible to keep the exsected heart in cold Locke’s
solution at ordinary room temperatures for hours and still to obtain powerful
and regular contractions of the heart when, after this procedure, it is
subsequently perfused with warm oxygenated Locke’s solution in the usual
way. Similar observations have been made on other contractile tissues and
will be dealt with in another communication.
In the meantime, it is sufficient to point out that those observations open
an easy way to experiments on a certain number of isolated human tissues,
removed for surgical reasons, by which experiments certain questions can be
answered which cannot readily, if at all, be decided in any other way.
So far as I am aware, this is the first time that pharmacological
experiments of this nature on isolated human tissues have been performed,
and by the simplification of technique dependent upon those observations on
the survival of involuntary muscle at ordinary temperatures, a field is open for
exact quantitative pharmacological experiments immediately upon those
tissues, whose reaction to drugs it is the final aim of pharmacology to deter-
mine. These experiments can be made under similar conditions to, and
therefore entirely comparable with, experiments made on tissues of those
mammals ordinarily used for pharmacological investigation.
One of the questions which require to be answered has regard to the
nature of the sympathetic innervation of the human uterus and its response
to certain drugs.
It has been shown by Langley and Anderson+ that the sympathetic
nerve supply to the uterus of the rabbit is motor in quality, whether the
uterus is in the pregnant or non-pregnant condition, and that adrenine has a
similar motor effect on it, On the other hand, it was discovered independently
by Cushny,t Dale§, and Kehrer|| that the uterus of the cat responds to
* Gunn, ‘Journ. of Physiol.’ vol. 46, p. 508 (1913).
+ Langley and Anderson, ‘Journ. of Physiol.,’ vol. 19, p. 122 (1895); Langley, zbid.,
vol. 27, p. 252 (1901).
t Cushny, ‘Journ. of Physiol.,’ vol. 35, p. 1 (1906).
§ Dale, ‘Journ. of Physiol.,’ vol. 34, p. 163 (1906).
|| Kehrer, ‘ Arch. fiir Gynikol.,’ vol. 81, p. 160 (1906).
552 Mr. J. A. Gunn. The Action of Certain
sympathetic stimulation or to adrenine by a motor effect when pregnant, but
by an inhibitor effect when non-pregnant; in the latter case, therefore
differing from the response of the rabbit’s uterus.
It has recently been shown* that a still different type of sympathetic
innervation holds good for the uterus of the rat and the guinea-pig, for in
those animals adrenine inhibits the uterine contractions both when the uterus
is pregnant as well as when non-pregnant.
There are, therefore, three known types of predominant sympathetic
innervation of the uterus in different species of animals, as shown in the -
following table :—
Reaction to Adrenine.
Non-pregnant uterus. Pregnant uterus.
TINK ocoeodapo caanabeoe Motor. : Motor.
OP a kocorangaoconc see UOOBEA Inhibitor. Motor.
Guinea-pig ............ Inhibitor. Inhibitor.
Now the type of innervation in the pregnant uterus of the rat and guinea-
pig raises the important question, namely, what is the quality of the
sympathetic innervation of the human uterus? Does it resemble that of the
rabbit, that of the cat, or that of the guinea-pig? This is obviously a question
of supreme importance in the use of adrenine or other sympathomimetic
substances in human labour, because if the pregnant human uterus is to be
relaxed by those drugs (as is the pregnant uterus of the rat or guinea-pig)
then the employment of them in inertia uteri or in post-partum hemorrhage
is not only valueless but definitely dangerous.
Though the kindness of Drs. Whitelocke and Dodds-Parker, Surgeons to the
Radcliffe Infirmary, Oxford, I have been able to obtain sufficient material to
satisfy at least part of this inquiry. The former gave me the uterus and a
Fallopian tube from one case, and Fallopian tubes from two other cases; and
the latter a Fallopian tube from one case. The organs were removed from
surgical necessities. As soon as they were removed, they were put into cold
Locke’s solution and conveyed to the laboratory. There they were put into a
bath of Locke’s solution, oxygenated and at a temperature of 37°, and the
movements recorded, the method employed being the now familiar method
used for isolated mammalian organs and first used for the uterus by Kehrer.
The isolated human Fallopian tube, when put into warm oxygenated saline
solution, almost immediately executes rhythmical movements, not in any
* Gunn and Gunn, ‘Journ. of Pharmacol.’ (1914).
Drugs on the Isolated Human Uterus. 553
decided way dissimilar from the contractions of the isolated uteri of ordinary
experimental mammals under similar conditions of experiment. Indeed the
readiness with which the human Fallopian tube passes into rhythmical
contraction makes it clear to me, after experience of other rhythmically
contractile tissues under the same conditions, that the Fallopian tube possesses
a high degree of spontaneous rhythmicity.
Several experiments which have been made have shown conclusively that
adrenine has a powerful motor effect on the human Fallopian tube.
The effect of adrenine is shown in fig. 1; in this case it produced a rise of
tonus with conversion of slower rhythmic contractions into more rapid
smaller ones.
Fig. 2 is shown because it illustrates (1) what I have found in four experi-
ments, the somewhat surprising fact that pituitrin has no pronounced effect
on the human Fallopian tube ; (2) because it shows the continued vitality of
the organ after it had remained for 30 hours in cold Locke’s solution, and the
still normal response to epinine.
That the vitality of the uterus is great under certain conditions of keeping
is a fact of which I was previously aware, because from unpublished experi-
ments made in this laboratory in conjunction with Dr. Hudston, it was found
that, after the guinea-pig’s uterus had been kept in Locke’s solution, at
temperatures of from 3 to 7° C., it still may execute rhythmic movements
when placed in warm oxygenated Locke’s solution, after having remained
quiescent at the low temperature for as long a period as seven days.
In regard to the uterus proper I have as yet obtained only one for experi-
ment. This uterus was removed from a patient, non-pregnant and about
40 years of age. The uterus was removed for disease of one Fallopian tube
along with a partial fibroid condition of the uterys itself, The other
Fallopian tube and part of the uterus was apparently healthy. The latter
was cut into strips and tested in the usual way.
The rhythmic movements of the uterine strips were much slower and more
infrequent than those of the Fallopian tubes.
Fig. 3 shows the effect of adrenine 1 in 250,000 on a strip of uterine muscle.
The strip had shown contractions lasting from about 30 to 60 seconds at
intervals of from 3 to 5 minutes. At the end of one of those contractions
" adrenine was added to the bath (fig. 3). This produced a strong tonic con-
traction with production of superimposed smaller waves. Other strips which
were tested gave a similar result.
It was interesting to compare, for further guidance, the effect of the same
concentration of adrenine on the Fallopian tube belonging to the same uterus.
Fig. 4 shows the result obtained.
‘QUIUOIPY JO Joo Ojo Surmoyg ‘sn104¢Q yuvuseid-uon ueumpP Jo di1z4g poze[os[—'g “OT
V¥
‘guiuidy Jo yo 1OjOW PU ULAVINGIG Jo Woo OANVSON SUIMOYG ‘TPAOCUIOL 109Je sAnoY Og ‘oqny, uvido][Ry WeUIN;, poxeposy—'Z “Hl y7
‘sBUI0eI} SUIMOT[OF PUB SITY UI Sprvadn poplodad UOTORIZUOD ‘oUTUAIPY jo oy Tojo Surmoyg ‘eqny, uvido][eq Wen, poyel[os]—'T “17
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it
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Mr. J. A. Gunn.
Drugs on the Isolated Human Uterus. 555
It is clear from these experiments that adrenine has a powerful motor
action on the non-pregnant human uterus and Fallopian tube. The presump-
tion is, therefore, that the sympathetic nerve supply to it is motor in quality.
Pituitrin decidedly stimulates the contraction of a strip of human uterus
as shown in Fig. 5. The effect is produced much less promptly than the
x
Fic. 4.—Isolated Human Fallopian Tube from same Uterus used in fig. 3.
i 5 ee eS
A Pituitrinlin 400 -
Rite Onet MUSE TMU me UN TUT NU Treen TUM TE NU Uenn nue rere
Fie. 5.—Isolated Strip of Human Non-pregnant Uterus. Showing Motor Effect of
Pituitrin.
effect of adrenine. No such pronounced effect could be obtained on the
Fallopian tube belonging to the same uterus, the addition first of pituitrin
1 in 800 and then 1 in 400 having no effect in 15 minutes, whereas the sub-
sequent addition of adrenine 1 in 250,000 produced immediately powerful
and enduring contractions., The negative effect of pituitrin on the tube is an
unexpected result, seeing that the musculature of the tube is continuous with
that of the uterus proper. It suggests that deductions cannot be drawn from
experiments on the Fallopian tube alone to embrace the uterus proper.
It has not been possible yet to secure for experiment a pregnant human
uterus, which is of course rarely removed. Unless, however, the human uterus
has a still different type of sympathetic innervation from that of all the
animals which have been examined, the probability is that adrenine will have
556 Prof. B. Moore. Presence of Inorganic Iron
a motor effect on the pregnant human uterus, and that, therefore, the use of
sympathomimetic substances in labour is justified.
Summary.
The movements have been recorded of the isolated non-pregnant human
uterus and Fallopian tube. Adrenine has a powerful motor action on both
these organs. The deduction is drawn that this is the qualitative effect of
sympathetic innervation of the human uterus, at least when non-pregnant.
Pituitrin also stimulates the human uterus proper to contraction, but no
such effect has been definitely obtained on the Fallopian tube.
The Presence of Lnorganic Iron Compounds in the Chloroplasts
of the Green Cells of Plants, considered in Relationship to
Natural Photo-synthesis and the Origin of Life.
By BenJaAMIN Moors, D.Sc., F.R.S., Professor of Biochemistry, University of
Liverpool.
(Received March 11,—Read April 30, 1914.)
(From the Johnston Biochemical Laboratory, University of Liverpool.)
It has been demonstrated by Moore and Webster* that colloidal solutions,
or suspensions, of salts or oxides of iron, in presence of dissolved carbon
dioxide and with the energy supply of sunlight, possess the power of synthe-
sising formaldehyde. Since this is known to be the first step in the process
of organic synthesis of the substance of all living plants and animals from
inorganic material and must, moreover, have occurred in past ages over
immense areas of the earth’s surface before life began to exist on the planet,
the conclusion was drawn that life must have originated by continual
development of more and more complex organic substances from this simple
commencement.
It is in this first stage of all that the greatest production of chemical
energy occurs, and accordingly a transformer of light energy into chemical
energy is essential. Although the more highly organised carbohydrates and
proteins still require catalysts for their synthesis, weight for weight they
contain scarcely any greater storage of chemical energy than formaldehyde,
* © Roy. Soc. Proc.,’ B, vol. 87, p. 163 (1918).
Compounds in Chloroplasts of Green Cells of Plants. 557
and once an accumulation of organic material has been reached, even the
fats and their allies can easily be synthesised by the combination of linked
exothermic and endothermic reactions by the living cell acting as a trans-
former, without the use of external energy such as that of sunlight.*
Once organic matter has been synthesised, the living cell can oxidise one
portion of this to reduce still more another portion. In this manner the
animal cell can oxidise carbohydrate, for example, and use the energy so set
free to build up another portion of carbohydrate into fat which weight for
weight contains double as much chemical energy as the carbohydrate, without
in the process making use of an external source of energy.
At the commencement, however, when only water and carbon dioxide are
the available materials, it.is indispensable that an external source of energy
such as sunlight should be available, and a suitable mechanism, or chemical
system, for the transformation of this store of energy into the chemical
energy of organic compounds.
Such a transformer has been recognised for a long period in the chloroplast
or chlorophyll-granule of the green cell of the higher plant.
Since the days of de Saussure,} now over a century ago, the green colouring
matter of the leaf, chlorophyll, has been regarded as the fundamental agent
for this world-wide photo-synthesis. But it is remarkable how completely
this view is based upon indirect or circumstantial evidence, and how little,
if any, direct observation can be cited in its support.
Chlorophyll is known by the biochemist to be one of the most complex of
substances, comparable to heemoglobin in its molecular structure, and yielding
a host of disintegration constituents themselves complicated substances of high
molecular weight. Between the simple colloidal molecules of inorganic iron
salts in solution or suspension and such a highly complex organic substance
as chlorophyll there is a wide hiatus, and it was with the view of discovering
some intermediate links or finding some explanation for the gap that the
present experiments were commenced.
Before describing the experiments in detail it is desirable to touch upon
present views as to photo-synthesis in the green cell as far as these bear upon
our investigation, in order to give an appropriate setting to the new facts, and
show how present knowledge regarding the absolute necessity for the presence
of iron in the green leaf, which has been hitherto devoid of all explanation,
led up to these experiments.
Photo-synthesis with production of oxygen only occurs in the chlorophyll-
* See Moore, ‘ Recent Advances in Physiology and Biochemistry, edited by Leonard
Hill, pp. 135, 138, Arnold, London (1906).
+ “ Recherches Chimiques sur la Végétation” (1804), ‘Ostwald’s Klassiker,’ Nos. 15-16
ViOln ioe ——B: 200;
558 Prof. B. Moore. Presence of Inorganic Iron
containing parts of the plant, and only in these when they are exposed to
light. Also, when a plant is allowed to grow in darkness, the leaves are
found to be pale-yellow in colour, or chlorotic, instead of green. When a
plant grown in darkness, and, as a result, possessing chlorotic leaves, is then
exposed to light, the pale-yellow colour is rapidly replaced by a green, and
then photo-synthesis is readily demonstrable by the evolution of oxygen and
the appearance of starch granules.
The above reasoning constitutes the whole of the evidence that chlorophyll
is the primary cause in the first act of photo-synthesis. It is to be observed
that the entire chain of evidence is inferential, and that in order to form a
valid proof, chlorophyll would require to be the only substance present in
the chloroplast, which is very far from being the case. No observer has ever
obtained an appreciable and satisfactory synthesis with pure chlorophyll in
solution or suspension when removed from the other constituents of the
chloroplast. Certain observers have observed minute traces of formaldehyde
formation with chlorophyll solutions or emulsions, but even these traces
of photo-synthesis have been stoutly denied by other competent observers.
In any case, the photo-synthetic effect produced is indattesia small
compared to that observed in the intact green cell.
The most recent and careful experiments upon this subject are those
performed by Usher and Priestley* and by Schryver.t Usher and
Priestley found that when a chlorophyll-containing extract from green
leaves was spread out as a film or emulsion on a gelatine plate, small, but
distinctly demonstrable amounts of formaldehyde were formed on exposure
to sunlight. But in this case there is gelatine and the inorganic colloids
it contains shown by its ash to be present, and in the chlorophyll extract
there would undoubtedly be iron salts present, because about one-fourth of
the iron of green leaves comes away in the alcoholic extract.
Schryver worked with an ethereal solution of chlorophyll allowed to
evaporate at room temperature on a strip of glass, and found that although such
films of chlorophyll on glass produced no formaldehyde in darkness even
in presence of moist carbon dioxide, a minute amount of formaldehyde was
formed when the film was exposed to sunlight even in absence of carbon
dioxide, and a distinct reaction when the film was exposed to sunlight
in presence of moist carbon dioxide. The amount of formaldehyde formed
in all such experiments is, however, very minute compared to the products
of photo-synthesis under natural conditions by the complete chloroplast.
* ‘Roy. Soe. Proc.,’ B, vol. 77, p. 369 (1906) ; B, vol. 78, p. 318 (1906) ; B, vol. 84, p. 101,
(1911).
+ ‘Roy. Soe. Proc.,’ B, vol. 82, p. 226 (1910).
Compounds in Chloroplasts of Green Cells of Plants. 559
Now the chloroplast contains a great deal more than chlorophyll, and when
all the chlorophyll has been removed by some such reagent as hot alcohol
there remains behind a colourless body, the so-called stroma. The chloro-
plast after the extraction is still a solid looking body, and to all
appearances the only thing that has happened is that a thin layer of green
colouring matter has been removed. There is no shrinking or shrivelling up
of the chloroplast.
There is accordingly no experimental evidence that the primary agent in
the photo-synthesis may not be contained in the colourless part of the
chloroplast, and the chlorophyll may be evolved at a later stage in synthetic
operations induced by some constituent of the colourless part. The function
of the chlorophyll may be a protective one to the chloroplast when
exposed to light, it may be a light screen as has been suggested by
Pringsheim, or it may be concerned in condensations and polymerisations
subsequent to the first act of synthesis with production of formaldehyde.
All these views and others are possible, and the function of chlorophyll in
the chloroplast remains for solution, but it has not been proved that
chlorophyll is the primary causative agent in the photo-synthetic process
where the chief energy uptake occurs with formation of formaldehyde.
There are other pieces of experimental evidence apart from the repeated
failures to obtain satisfactory synthesis with isolated chlorophyll which go
to indicate that chlorophyll is not the transformer in the first link of the
synthetic chain.
In the first place chlorophyll itself is a product of photo-synthesis, and
therefore there must be some active photo-synthetic substance present in
the chloroplast before the chlorophyll appears which indeed first produces
the chlorophyll by its activity.
When a yellow etiolated leaf taken from the darkness is exposed to the
light it contains no chlorophyll, but photo-synthesis, in the absence of
chlorophyll, sets in, and chlorophyll itself is one of the products, not the
originator or agent, of this photo-synthesis. ‘The period from first exposure
to light to the appearance of chlorophyll is too short to determine whether
oxygen production and starch formation commence before chlorophyll is
formed.
In the next place Engelmann,* by the application of his ingenious method
of the oxyphile bacteria has clearly demonstrated two important facts;
first, that the chloroplast alone, even when displaced from the rest of the
cell, can, in presence of light, go on synthesising and producing oxygen;
* ‘Botanische Zeitung,’ 1881, p. 446, and 1887, pp. 394, 410, 418, 426, 442, 458.
2uU 2
560 Prof. B. Moore. Presence of Inorganic Iron
and, in the second place, a still more important point in our chain of
evidence, namely, that certain leaves such as those of the yellow variety
of elder, which do not produce chlorophyll when exposed to light but
contain yellow chromatoplasts, cause synthesis and produce oxygen. These
observations ‘as to synthesis by healthy yellow leaves have been confirmed
by other observers such as Tammes, Josopait, and Kohl.*
The strongest piece of evidence, however, that iron salts are more funda-
mental to photo-synthesis and take an earlier share in it than chlorophyll,
is that furnished by that process frequently occurring in green leaves
known as “ chlorosis.”
Chlorosis is a pathological condition of green leaves of considerable practical
importance in arboriculture, and the discovery of its cause is, as Molisch
states, one of the most interesting and beautiful in the history of plant
physiology.
It was shown in 1845 by Eusebe Grist that chlorosis naturally occurring
in the leaves of shrubs or trees could be entirely removed either by
applying dilute solutions of iron salts to the roots, or by placing the
detached chlorotic branch in a dilute solution of iron, or even by painting
the chlorotic leaf with a very dilute solution of an iron salt. In some
cases within 24 hours, and in nearly all cases in a period of a week to
10 days, the green colouring matter developed in the leaves where none had
been before.
These results have been often confirmed and have been extended by
Salm Horstmar, A. Gris, and Sachs. Molisch§ has, moreover, shown in a long
series of experiments with different species of plants that all green plants,
even when fully exposed to light, become afflicted with chlorosis and fail to
develop chlorophyll when they are grown in a culture fluid especially made
devoid of iron. As soon as the reserve store of iron always contained in the
seed embryo and cotyledons has been exhausted in the primordial leaves, only
chlorotic pale-yellow leaves are formed. These pale-yellow leaves rapidly
turn green if minute quantities of an iron salt are added to the culture fluid,
or even if the surface of the leaf be painted over with a dilute solution of an
iron salt, as had been previously shown by Gris to be the case with
* Quoted by Czapek, ‘ Biochemie der Pflanzen,’ vol. 1, p. 447.
+ ‘De l’Action des Composés Ferrugineaux sur la Végétation,’ 1843. See also ‘Comptes
Rendus,’ vol. 19, p. 1118 (1844) ; vol. 21, p. 1386 (1845) ; vol. 23, p. 53 (1846) ; and vol. 25,
p- 276 (1847).
+ Salm Horstmar, ‘Versuche tiber die Ernahrung der Pflanzen, 1856; A. Gris,
‘Annales d. Scien. Nat.,’ Series IV, vol. 7, p. 201 (1857) ; Sachs, ‘ Flora,’ 1862.
§ Molisch, ‘Die Pflanzen in ihren Beziehungen zum Hisen,’ Jena, G. Fischer (1892).
Many of the references given are quoted from this source.
Compounds in Chloroplasts of Green Cells of Plants. 561
pathologically chlorotic leaves. So that iron is as indispensable to the green
leaf as it is to the red blood-corpuscle.
The remarkable thing, in view of this failure to develop chlorophyll in
absence of iron, is that chlorophyll itself is shown by all the more recent
researchers to be quite free from iron.* Chlorosis and its cure by iron
salts has accordingly remained a puzzle to plant physiologists ever since the
time of the discovery of Gris.+} The experiments to be recorded below
furnish, for the first time, a rational explanation of chlorosis and its cure.
The iron salts are necessary for the formation of the colourless portion of
the chloroplast, for when all the chlorophyll and other fatty bodies and
pigments are removed from the chloroplast by extraction with alcohol,
and the colourless chloroplastic residue is treated with the micro-chemical
tests for imorganic iron, a positive reaction in unmistakable degree is usually
given by the colourless residue of the chloroplast.
This inorganic iron in presence of sunlight must give rise to photo-
synthesis and production of formaldehyde which is then carried on into
sugar and starches by other constituents of the chloroplast, and it is
probably here, somewhere in the later processes, that the chlorophyll finds
its function. The chlorophyll itself, as shown by the facts of chlorosis, its
removal by administration of iron, and the presence of iron salts in the
colourless part of the chloroplast, is a product of synthesis from colourless
substances or from the light-yellow pigment. For the production of the
chlorophyll under normal conditions, both the presence of iron and the
energy of sunlight are essential.
The reason for the earlier erroneous view that the chlorophyll molecule
contained iron was that a certain fraction of the iron compounds contained
in the green leaf becomes extracted by the alcohol used in the first extrac-
tion of the leaf,t so that all crude chlorophyll extracts contain iron. This,
however, disappears on treating the alcoholic extract with benzol, and the
product of purer chlorophyll separating from the benzol fraction is iron-
free. At the same time its spectrum and other physical properties prove it
to be unaltered chlorophyll.
Other facts which show the importance of iron compounds in the green
leaf are that leaves which are not deciduous annually, such as pine needles,
contain more iron in their later years, and also in leaves of annual growth
* See Molisch, (oc. cit., and R. Willstatter u. A. Stoll, “ Untersuchungen itiber Chloro-
phyll, Berlin, J. Springer (1913).
+ See Czapek, ‘ Biochemie der Pflanzen,’
{ According to early observations of Boussingault ( Agronomie,’ vol. 5, p. 128) from
one-quarter to one-third of the iron is removed by the alcohol.
562 Prof. B. Moore. Presence of Inorganic Iron
the older the leaf is the more iron does it contain in its ash. Thus
Boussingault found in the ash of young leaves of Brassica 2 per cent. of
Fe203, while old leaves contained in their ash 9°64 per cent. Lactuca sativa
had in the young leaves 2°67, and in the old leaves 6:43 per cent. of FesO3
in the ash. Another point is the curious conservation, resembling that seen
in the animal economy, of the iron of the leaf in the case of deciduous
leaves. Before the leaf drops a good deal of the iron is re-absorbed and stored
for future use. This is shown by analyses of the iron of the leaves of
Fagus sylvatica made by Rissmiller* in successive months. The figures
quoted give quantities of Fe203 in 100 parts of dried leaves collected at the
times of year stated—
May. June. July. Aug. — Sept. Oct. Nov.
Oxide of 1roniees-se: 035 O51 058° 075 1:03 “0:60 aise
The gradual increase of iron content to a maximum followed by a fall as
the leaves grow sere is very interesting. ;
It has been shown by Molisch (oc. cit.) that iron is an essential constituent
for the growth of all plants, whether green or otherwise, but the saprophytic
and parasitic plants which contain no chlorophyll require much less iron
and, as a rule, contain much less in their ash. Our own experiments show
that the histo-chemical reactions for iron develop much more slowly in the
fungi and are much less intense in degree. These feebler reactions probably
arise from organic compounds of iron slowly being decomposed in traces and
setting free ionic iron. These organic iron compounds of the fungi are
concerned with some other function than photo-synthesis or chlorophyll
formation ; they probably take a part in nuclear structures, for many nucleins
are iron-containing, and, as has been shown by Macallum, after treating with
acid alcohol to unmask the iron previously present in an organic form, the
chromatin of nuclei always contains iron.
The reactions for inorganic iron are shown most markedly with the more
lowly organised plants such as unicellular green plants occurring alone or in
lichens, or in delicate algal threads, but when proper precautions are taken
they can also be clearly demonstrated in the chloroplasts of the higher
plants. The reactions are particularly well shown by the chloroplasts of
aquatic plants, where, as is well known, the percentage of iron in the ash is
also high.
These facts are in keeping with the natural order of evolution and are
also in accord with other observations. For example, many alge (such as
* “Ueber die Stoffwanderung in der Pflanzen,” ‘Landw. Versuchsstationen, 1874
(‘Just’s Jahresbericht,’ vol. 2, p. 849, 1874).
Compounds in Chloroplasts of Green Cells of Plants. 563
- certain Confervee and Cladophora) deposit around them a layer of yellow to
rust-red colour consisting of mixed ferrous and ferric oxides; this is often
actively secreted from waters containing only traces of iron.
A considerable number of lichens also secrete incrustations of the mixed
oxides of iron to such an extent as to change their appearance to an iron-
oxide or ochre colour, so that they have been termed by systematic botanists
“forme oxydatze, ochracez ” or “iron lichens.’ The iron-oxide forms a fine
incrustation usually on the mycelium of the fungus. No association of this
iron-oxide with a photo-synthetic function has ever been suggested, but in
view of our present knowledge of the photo-synthetic activity of iron salts
some investigation in this direction is highly desirable. It is an interesting
observation of Molisch, from our point of view, that these “iron lichens”
flourish exclusively on the oldest primitive rock-formations (‘ Urgestein ”).
They are never found upon chalk formations, but grow upon granite, gneiss,
syenite, and porphyry. Molisch was unable to find inorganic iron in the
other lichens, but this doubtless arose from the less delicate methods he had
at his command at that time, and from the fact that the fatty bodies con-
tained in the green cells of the alga of the lichen had not been removed.
When the lichen is extracted with alcohol and Macallum’s hematoxylin test
then applied, the algal cells rapidly stain a deep blue-black, showing the
presence of inorganic iron, while the hyphe of the fungus only take on a
brownish tinge during the same time, and only give a faint positive reaction
abt the end of some days or weeks.
It is somewhat remarkable that the presence of iron in the chloroplast
should for so long have escaped discovery. The explanation probably lies in
the fact that little attention has been given to the application to the green
cell of the histo-chemical tests for iron since the discovery by Macallum of
the more delicate hematoxylin iron test, as also to the delicacy of the
chloroplasts to the more drastic earlier method used by Molisch, and to these
factors may be added the difficulty with which some of the chemical reagents
for iron penetrate the green cell, and the presence in the chloroplast itself of
fatty and lipoidal substances which prevent the ingress of the water-soluble
stains.
Macallum* in 1894 before his discovery of unmordaunted hematoxylin as
a reagent for iron, and using then ammonium sulphide in glycerine as a
reagent, states that bacteria gave no evidence of an organic iron compound,
but in the Cyanophycee the chromophilous portions of the “central
substance” contain iron, and iron may be also demonstrated in the peripheral
granules containing the so-called cyanophycin. At this period, Macallum
* *Roy. Soc. Proc.,’ vol. 57, p. 261 (1894).
564 _ Prof. B. Moore. Presence of Inorganic Iron
was specially concerned in proving the presence of organic iron in the
chromatin of the nucleus and was not searching for iron in the chloroplasts,
so that the reference above to the presence of iron in the cyanoplasts of the
Cyanophycee is highly interesting to-day.
Molisch (/oc. cit.) used long immersion in satwrated potassium hydrate as a
preliminary method for setting free masked iron («.e. organic iron) in available
form for after-detection by potassium ferrocyanide and hydrochloric acid, and
in the later testing used very strong hydrochloric acid (10 to 20 per cent.).
Such drastic procedures are very dangerous, because the alkali breaks down
the delicate chloroplasts, and may also itself contain iron salts in traces; also
in the second place, as pointed out by Quincke,* such strong acid will fairly
_rapidly set iron free in inorganic or ionic form from the potassium ferro-
cyanide reagent, and this ionic iron reacting with the remainder of the reagent
will give the Prussian blue colour. Molisch found more iron in the epidermis
and fibro-vascular bundles of green leaves than in the green mesophyll, but
as he himself admits “ the potassium hydrate so disorganises the nucleus and
chlorophyll-granules that one can conclude nothing as to the distribution of
iron in the cell.”
So far as we have been able to discover there exist no records later than
the above in the literature of the subject on the occurrence of iron in the
chloroplasts of the green cell, nor any information as to the form in which
iron compounds are present. No investigations with the iron hematoxylin
test of Macallum appear to have been made hitherto upon plants.
Expervmental Methods.
In carrying out tests for the detection of inorganic iron in the chloroplasts,
and in plant tissues generally, two points must be carefully borne in mind,
first, the previous preparation of the tissue and its subdivision so that the
parts possibly containing iron may be penetrated by the reagents used for the
detection, and secondly, that the reagents be applied carefully so that false
results are not obtained. Here care must be taken with the concentration of
the reagents and the relative periods of time within which positive results
are obtained.
In regard to the preparation of the tissues, if sections are to be cut, care
raust be taken that this is done with a clean burnished knife. Control
experiments show that a clean steel knife leaves no iron on the section. But,
in most cases, since the question at issue is not the structural arrangement
but rather whether this or that constituent contains iron, it is better to work
with finely teased or broken up tissues. For this purpose glass rods drawn
* “Arch. f. Exp. Path. u. Pharm.,’ vol. 37, p. 183 (1896).
Compounds in Chloroplasts of Green Cells of Plants. 565
out to a point were always used instead of steel needles, and also, in order to
break up some of the green cells and set free the chloroplasts, a portion of
the tissue in each case was still more broken up by turning upon it the blunt
end of the glass rod and grinding it between this and the microscope slide on
which it was being mounted.
In choosing tissues for examination, preference so far as possible is given
to those where the chloroplasts are more conspicuous in size, and also in some
cases, such as Spirogyra, delicate filaments were chosen which could, after
extraction as described below with alcohol, be mounted without breaking up.
In certain cases, such as Plewrococcus, staining can readily be obtained
without previous chemical preparation of the tissue, but, in the majority of
cases, the lipoids present along with the chlorophy!] in the chloroplast prevent
the penetration of the stain, also the green colour modifies and masks the blue of
the hematoxylin in Macallum’s test. For this reason itis well to remove the
lipoids and chlorophyll, and in many cases this is by no means an easy task.
In some cases standing in cold aleohol removes the chlorophyll quite effectually
and leaves the tissue colourless and ready for staining ; but in other cases the
tissue may be left at ordinary temperatures for days in alcohol, and this may
even be followed by several extractions with ether, and still some of the
green colour remains. After a good deal of experimentation the best
extractive in these latter cases was found to be boiling alcohol.
The tissue, either partially teased with the glass points if it is bulky lke
the leaf of a higher plant or a piece of lichen or moss, or left intact if
a delicate structure like an algal filament or Plewrococcus, is placed in water
in a watch-glass and then absolute alcohol is gradually added portion-wise
and pouring away excess of the mixture at intervals until the fluid is finally
all absolute alcohol. The preparation is then boiled in the alcohol and the
greenish extract poured away, and this is repeated till the green tissue
becomes colourless. The decolorised tissue is then brought back again into
distilled water by gradually adding the water to the alcohol, and pouring off.
Finally, it is allowed to stand a few minutes in a watch-glass in water
redistilled from glass, and is then ready for staining.
In addition to the unmordaunted simple aqueous solution of well-washed
hematoxylin in $-per-cent. concentration introduced by Macallum* the
older histo-chemical tests for iron were also utilised, namely, potassium
ferrocyanide and hydrochloric acid for ferric salts, potassium ferricyanide
and hydrochloric acid for ferrous salts, and ammonium hydrogen sulphide in
glycerine for both. In our opinion the Macallum test surpasses all these in
reliability and delicacy. Its only fault is that it is too delicate, and the
* ‘Journ. Physiol.,’ vol. 22, p. 92 (1897).
566 Poe B. Moore. Presence of Inorganic Iron :
small traces of inorganic iron set free from organic compounds in the tissues
on long standing cause faint but increasing staining when a preparation is
left over for some days. When a blue-black is obtained, however, within
a few hours with this reagent it is a decisive proof of loosely combined, or
inorganic, iron in the situation where the staining occurs.
Ammonium hydrogen sulphide when added to the tissues with an equal
amount of glycerine, and the whole kept at 36° C. for some hours, produces
a distinct blackening as compared with the normal, but the effect is not
very pronounced and is only clear on comparison of treated and untreated
tissue.
Potassium ferroeyanide and hydrochloric acid never gave a blue colour,
but a blue colour is frequently, and very distinctly, given within a few hours
by potassium ferricyanide and acid, demonstrating that the inorganic iron of
the chloroplasts is present in the ferrous condition; this was typically
observed in the case of Spirogyra.and Vaucheria.
There is always some doubt, however, about using a reagent which itself
contains the element sought for, and moreover is fairiy readily broken down
in presence of organic matter and acid.
The hydrochloric acid used should not exceed 0°5 per cent. in concen-
tration, and be used in equal volume with the 1:5-per-cent. ferricyanide
solution so that the concentration of acid acting on the tissue is only
0:25 per cent. Then, if a blue stain is obtained with a considerable intensity
within 24 hours, it may fairly certainly be attributed to ferrous iron in that
particular situation. The result, however, ought always to be confirmed by
the Macallum test, for solid starch or casein left for 24 hours in contact with
the above reagents each give a faint blue colour which increases as the
mixture is left standing.
When a solution of hematoxylin in pure distilled water is mixed with
a very dilute solution of an ordinary iron salt such as ferric chloride, a deep
blue-black coloration is immediately produced. If, instead of an ordinary
iron salt solution, a solution of highly colloidal or dialysed iron oxide be
mixed with the solution of hematoxylin there is obtained instead a deep
chocolate-brown coloration. In the course of some hours to a day or two,
this chocolate brown is replaced by the blue-black colour obtained with
ionic or crystalloidal iron salts. Similar results are usually obtained when
the hematoxylin solution is used as a detector of iron in the tissue of plants.
In certain cases, notably unicellular green plants and algal filaments, a deep
blue-black is obtained within a few minutes without any previous appear-
ance of the brown stain characteristic of colloidal iron oxide, while in many
of the higher plants (mono- and dicotyledons) the green leaf at first stains
Compounds in Chloroplasts of Green Cells of Plants. 567
a deep brown, which gradually, in a varying period of a few hours to a day
or two, changes to a blue-black, just as is seen in the test-tube when
colloidal iron oxide solution is mixed with the reagent. In certain cases,
however, the brown colour is found to persist for weeks without change.
This deep brown coloration is not simply due to imbibition of the tissue
with unaltered hematoxylin, for it is far too deep for this, and, moreover, is
not removed by washing with a mixture of equal parts of alcohol and ether
as recommended by Macallum. It is a true staining of colloidal iron,
present in those parts of the tissues where the brown occurs, and possesses
just the same dark brown colour that is obtained on mixing colloidal iron
oxide solution and hematoxylin.
In contrast with vegetable tissues, such a direct staining (either brown or
blue-black) is only found in the embryonic condition in the tissues of higher
animals, for the iron in the majority of such animal tissues is firmly bound
organically and gives no coloration with hematoxylin.
It is to be remarked that this staining as a test for iron is quite different
from the ordinary use of hematoxylin as a nuclear stain in histological
technique. In the ordinary use of hematoxylin as a staining reagent
a mordaunt is always used either preceding the hematoxylin, as, for example,
the iron alum mordaunt for Heidenhain’s iron-hematoxylin method, or
simultaneously as in the use of the hemalum stain, where the mordaunt
alum is mixed with the hematoxylin. But in Macallum’s use of the stain
no mordaunt whatever is used, but instead a simple aqueous solution in
pure distilled water. This solution only strikes a colour where a mordaunt
is naturally present in the tissue. Now with iron in colloidal form the
colour struck is the deep brown mentioned above, with iron in crystalloidal
form the colour struck is blue-black. Thus Macallum’s method resembles
Heidenhain’s staining, but with the previous iron treatment naturally
provided in the tissues, and the blue-black effect obtained closely resembles
in many cases a Heidenhain iron-hematoxylin stain.
In order to use the method effectively, it is not merely necessary to avoid
all minute traces of iron in the water and other fluids used, but also all
traces of alkali and acid, since these interfere with the delicacy of the
reaction. Alkali gives a rose-red colour with the hematoxylin, and acid
inhibits the development of the blue-black when the amount of iron is
small. In making up the stain itself, water twice distilled from glass
vessels must be used as the solvent, the second distillation having been
made immediately previous to use. To make the staining solution, 0°3 grm.
of pure hematoxylin is weighed out, and washed with the twice distilled
water till the crystals are colourless, and the wash-water is only pale
568 Prof. B. Moore. Presence of Inorganic Iron
yellow without any trace of red. The solution is then made up to 50 C.0.
and kept in a Jena glass flask, for the alkali which is slowly dissolved out
from ordinary glass rapidly turns the solution pink. The reagent should be
pale yellow when used, in order to obtain the best effects, and does not keep
in good condition for more than a few days.
After the chlorophyll and fats have been removed from the tissue by
allowing it to stand in cold alcohol, or by boiling up with alcohol, the
- colourless tissue must be well washed with water, and the water used must,
as described above, be doubly distilled from glass.
The staining process may be watched in progress, when it will be found
that escaped chloroplasts from ruptured cells take on the stain first, and in
many cases show a deep purple-blue within a few minutes. Within the
intact cell the stain does not penetrate so rapidly, and the cell wall may
show a blue staining in some cases before the contained chloroplasts, but
eventually these also stain a deep blue, sometimes preceded by a dark
brown. The nuclei of the green plant cells also stain a deep blue (unlike
animal cell nuclei), and there is usually a much slighter diffuse blue in the
remaining cytoplasm. The fibres associated with the vascular bundles also show
in many cases a blue staining. This probably means that the iron salts are
carried along this route to the green cells. But the early and deep staining
of chloroplasts and nucleus are characteristic in the preparations.
In addition to tissues containing chloroplasts, several preparations have
been made from plants not containing chlorophyll, such as yeast, moulds,
and larger fungi. There is a marked contrast found here, a blue stain does
not appear for some days, and then in only a comparatively feeble manner.
The conidia and the conidiophores show more iron than the mycelium filaments.
It is probable that this slow and feeble staining is due to organic iron
compounds slowly breaking up and yielding traces of inorganic iron.
A series of ash analyses of chlorophyll-containing and chlorophyll-tree
plants show in all cases a much higher percentage of iron in the ash of the
green plant; these analyses will furnish the subject of a separate paper.
A large number of plants of different types have been examined, and the
main results are given in the following account.
Amongst unicellular green plants there were examined Chlorella, obtained
as plankton from a green-coloured pond water; Plewrococcus, obtained ‘in
nearly pure condition growing on an oak fence near Oxford, and stained and
examined in collaboration with Mr. Edward Whitley; several forms of
diatom and several unicellular forms found in lichens.
The blue-black effect is very readily obtained with these unicellular green
plants, often without previous removal of the chlorophyll. In the case of
Compounds in Chloroplasts of Green Cells of Plants. 569
the lichens the contrast is marked between the green cells and the fresh
hyphe of the fungus, but dead or decaying fungal matter often gives a
blue stain.
The alge observed were species of Vaucheria, Spirogyra, Ulva, and
Ulothriz. The effects were often repeated in several experiments, both with
hematoxylin staining and with ferricyanide and hydrochloric acid. The
ferricyanide solution does not appear to penetrate well, and only some
filaments in an alga like Spirogyra are coloured, but the staining has been
obtained within an hour or two of treatment with this reagent, and is a very
beautiful effect when obtained in Spirogyra. The light blue colour follows
the spirals of the chlorophyll bands, and the granules are obviously more
deeply blue than the rest of the bands. The deep blue-black with -
hematoxylin is more readily and uniformly obtained, coming often within
a few minutes of applying the stain to the decolorised alga, and furnishing
again a beautiful effect. Sometimes, however, the brown colour of colloidal
iron is obtained in Spirogyra.
Ulva latissima gives a very deep blue-black coloration, rendering the cells
almost opaque ; its ash shows a high content in iron.
Cladothrixz, when growing in water containing small amounts of iron, as is
well known, secretes, or excretes, a tube of iron oxide around the filaments,
and is then known as an “iron bacterium.” When these so-called “iron
bacteria” are treated with hematoxylin, they turn blue-black almost
instantly, and, if the stained specimens are examined under the microscope,
the interesting fact is immediately observable that not only the external
tube, but the substance of the organism itself, is stained blue-black, so
setthng a much disputed point. The same is seen in Vaucheria, an
incrustation of iron-oxide particles is demonstrable in the gelatinous sheath
surrounding the filaments, either by ferricyanide or hematoxylin staining,
but, in addition, both reagents show inorganic iron within the filament itself.
Many higher aquatic plants, such as Lemna and Hlodea, possess such
incrustations of iron oxide on their leaves when grown in water containing
only traces of iron, but in such cases it is also found that the chloroplasts
of the green cell itself are very rich in inorganic iron. The higher aquatic
plants examined have been these two and a variety of water-cress, and all
three were found to give a strong positive reaction.
Ordinary lawn grass contains a high percentage of iron in the ash, and,
when teased out and deprived of chlorophyll by hot alcohol, forms a very
suitable object on account of the ease with which strands of fibre with
attached cells separate. The staining of the chloroplasts is at first a dark
brown passing later into a blue-black. The leaves of many species of
570 Presence of Inorganic Iron Compounds in Chloroplasts.
dicotyledonous plants were examined and it was found that here the
transition from dark brown to blue-black was much slower as a rule, and
in some cases the staining remained permanently of a deep orange-brown
to a pure dark brown colour. But in all cases the chloroplasts stained
more deeply than the remainder of the cytoplasm.
The catalyst for the photo-synthesis may not in all cases be an iron salt,
or oxide, but an iron salt is present and capable of operating as a catalyst
in a large number of instances.
Various substances known to be present in the ash of leaves have been
tested for their photo-synthetic activity in connection with the work, and it
has been found that magnesium and calcium phosphates and bicarbonates are
entirely ineffectual, but that marked photo-synthesis of formaldehyde is
obtained with chlorides or colloidal hydrates of iron or aluminium.
Summary.
1. Inorganic iron salts and iron or aluminium hydrates in colloidal solution
possess the power of transforming the energy of the sunlight into chemical
energy of organic compounds.
2. Inorganic iron, in erystalloidal or colloidal form, is present in the
colourless part of the chloroplast of the green plant cell in many plants.
3. In the absence of iron the green colouring matter cannot develop in
the leaf, although the green colouring matter itself contains no iron.
4. In the presence of sunshine, the iron-containing substance of the
chloroplast develops the colouring matter, so that this itself is a product
of photo-synthesis induced by the iron-containing compound.
5. These facts afford an explanation of chlorosis, and its cure by inorganic
iron salts, and demonstrate that iron is a primary esseutial in photo-synthesis,
and the production of chlorophyll.
6, The iron-containing substances of the colourless portion of the chloro-
plast, and the chlorophyll produced by them, then become associated in the
functions of photo-synthesis as a complete mechanism for the energy trans-
formation.
My thanks are due to my colleague, Prof. R. J. Harvey Gibson, for much
valuable advice in selecting and obtaining suitable material, and to Misses
E. M. Blackwell, M. Knight, and R. Robbins, of the Botanical Department of
the University of Liverpool, for supplies of fresh material.
571
Office Libra
Croontan Lecture: A New Conception of the Glomerular
Function.
By T. G. Bropiz, M.D., F.R.S., Professor of Physiology in the University
of Toronto.
(Lecture delivered June 15, 1911,—MS. received December 9, 1912.)
[PLATE 26.] ;
I have chosen as the subject of this lecture the physiology of the kidney,
and more particularly the mode of action of one part of it, namely the
glomerulus. In 1906, at the meeting of the British Medical Association in
Toronto, I brought forward a new conception of the action of this very
characteristic portion of the renal apparatus, and since that time have been
accumulating a considerable mass of evidence by the light of which my theory
can be criticised.
Very shortly after the discovery of the main details of the structure of the
_ kidney. Ludwig, basing his ideas upon the then known structure, put forward
his well-known theory that the glomerulus was a filter, and since that time
all discussions upon renal activity have centred round this theory because it
offered an explanation of the mode of action of one part of the mechanism
" upon hydrodynamic principles. The necessary corollary following from this
assumption of filtration is that a considerable degree of absorption must be
effected as the dilute filtrate travels down the tubule, and how excessively
great this must be was first pointed out by Heidenhain.
If we consider the results obtained by the earlier workers upon the kidney,
very many of them appear sufficiently well explained by the Ludwig theory,
but as in the course of years a far stricter examination of the theory was
attempted, several observations were made which proved very difficult to
explain, and in many cases it was necessary to make such extensive and often
contradictory assumptions that it became increasingly difficult to accept the
theory. Of recent years evidence has been obtained in many directions
which in my opinion conclusively proves that the glomerulus is not a
filtering surface. It is not my object to-day to discuss this point in any
detail. I may refer to my lecture delivered before the Harvey Society in
New York in December, 1909, where a short summary of the facts for and
against filtration is given, or to the excellent paper by Magnus, in the
‘Handbuch der Biochemie, where it is discussed in eztenso. It will be
sufficient for my present purpose if I indicate the chief reasons which led me
VOL. LXXXVII.—B. Pe 3
EY (ye | Prof. T. G. Brodie.
to conclude that the idea of filtration at the glomerular surface must be
abandoned. ‘
Perhaps the most striking piece of evidence is derived from the considera-_
tion of the concentration and constitution of the urines obtained during
extremely free secretion. The evidence is quite clear that the main bulk of
the water secreted by the kidney undoubtedly comes from the glomeruli.
Hence the more rapid the flow of fluid from the kidney the more closely
must that fluid resemble in constitution the fluid discharged from the
glomeruli, since a much shorter time is then allowed to the cells of the
tubules to modify it by absorption or secretion, and if filtration is the active
process in the glomeruli this fluid ought to approximate more and more
closely in composition to the blood plasma so far as the salts, urea and all
constituents of the plasma other than proteins are concerned. But the dilute
urine secreted after drinking copious amounts of lager beer,* or of water,>
shows a constitution in salts widely different from that of the blood. Con-
sidering only the total concentration, as estimated by the depression of the
freezing point, it is quite easy to obtain a urine with A=—01° C., and one
as low as—0°075° C. has been recorded.t To effect a change in concentration
so extensive as this denotes, by filtration through a semipermeable membrane,
would necessitate a pressure difference on the two sides of the membrane of
at least 4000 mm. Hg, a pressure difference utterly out of comparison with
the blood-pressure. Therefore to make such a result accord with the filtration
theory, it becomes necessary to assume a most extensive reabsorption of
the salts and other substances of small molecular size, a reabsorption on such
an extensive scale and at such a rate as is, I think, entirely out of the
question. :
If, in the second place, we investigate the correlation between the blood flow
and the rate of secretion, we find that while there is a general correspondence,
in that increased urine flow is usually accompanied by increased blood flow,
this is by no means a universal rule.§ Ihave frequently observed in kidneys
in which there was at the start a fairly free blood flow and but slow urine
secretion, a copious diuresis to come on without any change in the blood
flow. Indeed on no less than five occasions I have seen a distinct decrease
in the blood flow to occur as the diuresis commenced, and moreover in these
experiments the volume of the kidney actually increased. In every direction
we find that the urine flow does not vary strictly with the blood flow nor
* Dreser, ‘Arch. Exp. Path.,’ 1892, vol. 29, p. 303.
+ Macallum and Benson, ‘ Journ. Biol. Chem.,’ 1909, vol. 6, p. 87.
t Macallum and Benson, loc. cit.
§ Cf. Gottlieb and Magnus, ‘ Arch. Exp. Path.,’ 1901, vol. 45, p. 228.
A: New Conception of the Glomerular Function. 573
with the blood-pressure, as should be the case were filtration the essential
factor in determining the volume of the urine discharged from the kidney.
In the third place we have very decisive evidence against the Ludwig
theory in experiments designed to test the second assumption in that theory,
namely that of reabsorption. If this is a process which occurs extensively
within the tubules, and we bring into play any factor which favours
reabsorption, we ought to effect a diminution in the volume of
urine yielded by the kidney. Such a factor is an increase in hydro-
static pressure within the ureter, tending to prevent the outflow of
urine. (All that is necessary is to make the kidney discharge against a
small pressure. The experiments carried out by most experimenters
upon these lines have indeed yielded results which may be interpreted as
indicating increased reabsorption. But we may urge as a general criticism
against such results that the degree of decrease of urine flow is surprisingly
small when we remember how essential it is according to Ludwig’s theory to
assume that reabsorption is excessively free. The kidney working against
even a small hydrostatic pressure ought to show far greater reabsorption than
was actually obtained. But the whole idea of reabsorption as an active pro-
cess in the formation of urine has been completely disproved by Miss Cullis
and myself,* for we were able to prove that decrease in rate of the urine
flow when a kidney was made to secrete against a pressure was only a
universal result when the animal was under an anesthetic, and that if the
animal were pithed and the experiment then performed in the absence of
an anesthetic, the kidney working against a small pressure always excreted
more salt and usually more water than the opposite kidney.t The action of a
‘pressure then tends to excite the kidney to greater activity, a result which
entirely disproves the possibility of reabsorption being an extensive factor
in the normal formation of urine.
Yet another point which militates greatly against the idea that the
glomerulus is a filter is the behaviour of the kidney after temporary
asphyxiation. If the renal artery be clamped for one minute and then
released, the kidney does not at once begin to secrete, although the blood
flow returns at once. It is only after a variable, but usually considerable
delay that the kidney restarts, and at first the urine flow is very slow,
only gradually returning to a rate comparable to the initial flow. If the
artery has been clamped for any length of time the urine first collected after
* Brodie and Cullis, ‘Journ. of Physiol.,’ 1906, vol. 34, p. 224.
+ Subsequent to these experiments I have found that, under the same conditions, the
blood flow through the kidney is not altered by the small rise in ureter pressure employed
in our experiments.
Zin Xen
574 Y upegey Glipoia
the re-establishment of the circulation contains protein, casts, even hemoglobin,
indicating considerable damage to the renal epithelium, either of the tubules
or of the glomeruli or of both. But even if the glomerular epithelium be
damaged it is inconceivable that this should temporarily abolish all the
filtering properties it formerly possessed, and it is just as difficult to under-
stand why the recovery of its power to filter should occur so gradually when
the asphyxiation is arrested.
Let us next turn to the evidence that has been sought in favour of Ludwie’s
theory from experiments upon the maximum ureter pressure. One of the
earliest attempts to associate the formation of urine directly with the blood-
pressure was a measurement of the maximum height to which the kidney
could force the urine up a vertical tube. As is well known, in the case of
the salivary gland, the gland can secrete water to a pressure exceeding that
of the blood in the carotid artery, a clear indication that a new force,
viz., one exerted by the salivary gland cells, is at play in producing the
result. But in the case of the kidney the result is very different. For the
maximum ureter pressure always lies below the aortic blood-pressure, and
usually some 30-40 mm. Hg below that pressure. The results were therefore
interpreted by supporters of the filtration theory as indicating that as soon
as the pressure within Bowman’s capsule reached a point some 30 mm. Hg
below the glomerular blood-pressure, filtration ceased, and Starling* explained
the difference between the aortic pressure and the maximum ureter pressure
as being the pressure difference necessary for the separation of the blood
proteins from plasma, for he estimated the osmotic pressure of the blood
protein at that amount. It has since been shown, however, that the protein
osmotic pressure is certainly much less than this. Moreover Starling failed
to allow for a loss of pressure between the aorta and the glomerular
capillaries. Without doubt the loss of pressure between these points is less
than in the case of ordinary capillaries, tor the resistance in the kidney arterioles
when dilated is certainly much less than at most points on the systemic
circulation. As I shall show later, the maximum ureter pressure as ordinarily
taken is a measure of the blood-pressure in the glomerular capillaries.
But a still more difficult problem is offered to those accepting the filtration
theory in explaining these experiments. As was first pointed out by
Heidenhain,t upon the Ludwig theory the maximum ureter pressure should
be that pressure which just suffices to effect complete reabsorption of all the
glomerular filtrate. Upon the theory we are to imagine an absorbing surface,
capable of absorbing water, chlorides, urea and most of the bodies filtered in
* Starling, ‘Journ. of Physiol., 1899, vol. 24, p. 317.
+ Heidenhain, ‘ Hermann’s Hdb.,’ vol. 5, p. 327.
A New Conception of the Glomerular Function. 575
urine at a very fast rate. Such an absorbing surface would be influenced, as
indeed is usually assumed by the supporters of Ludwig’s theory, by a rise in
pressure of the fluid at the surface. It then becomes very difficult to explain
how the ureter pressure could ever be driven so high as is usually observed,
especially when we remember that the rise in pressure can be effected with
great rapidity.
Yet another result obtained in these experiments upon maximum ureter
pressure is very significant. I have found that the maximum ureter pressure
is practically the same whether the kidney be made to secrete a moderate
amount of urine or a very large quantity. If reabsorption be a very active
process, then the maximum ureter pressure in the latter case ought to be
distinctly higher than in the former. As a matter of fact, it is not.
Taking everything into account, therefore, I have very grave doubts as to
the occurrence of reabsorption in the tubules, and I am sure, if it does take
place, that it is insignificant in comparison to that demanded by Ludwig's
theory.
The Function of the Glomerulus.
Arriving then at the conclusion that the filtration theory was incorrect, I
came back once more to the old problem: How are we to explain the very
peculiar and characteristic structure shown by the glomerulus? I finally hit
upon the idea that it was simply a means of utilising the blood-pressure for
setting up a pressure head sufficiently great to drive the urine secreted at
the glomerular surface down the tubule. To express this idea I term the
glomerulus a propulsor. As is abundantly proved, the main volume of the
water of the urine is secreted into the capsule of the glomerulus. To drive
it from the capsule down the tubule requires a definite pressure-head.
_ Whence is this pressure head derived? My view is that the intraglomerular
blood-pressure is transmitted directly through the thin-walled glomerular
loops to the fluid which has been secreted into the capsule, and thus a
pressure is communicated to the fluid sufficient to force it down the tubule.
To test this view, let us imagine that a certain amount of fluid has accumu-
lated within Bowman’s capsule. The problem then becomes: How is that
fluid discharged down the tubule? If we know the number, length and
lumina of the tubules, and the total amount of fluid leaving the kidney within
a given time, it becomes easy to calculate the pressure-head which must
have existed within each capsule in order to drive the fluid out of the kidney.
It is simply an application of Poisseuille’s law. I therefore performed two
experiments upon the following lines. An active diuresis was established
in an anesthetised dog, and the rate at which urine was being discharged
from one of the kidneys was determined. The pedicle of the kidney was
576 Prof. T. G. Brodie.
then ligatured and the kidney fixed entire in 10-per-cent. formalin solution.
After fixation the whole kidney was cut into slices each about 7 mm. thick.
The medulla was carefully separated from the cortex, and the latter collected
and weighed. Next three small pieces of the cortex, selected from
different regions of the kidney, were weighed separately. These were
imbedded in paraffin and serial sections mounted. The sections were about
8 thick, The next point was to determine the number of sections through
which a single glomerulus extended. For this purpose ten glomeruli were
followed through the series, and the mean number of sections through which
one glomerulus ran thus ascertained. Lastly the total number of glomeruli
in each section was counted, and the total number for all sections, divided by
the average number for a single glomerulus, gave the total number of
glomeruli present in that block of cortex. Similar calculations were made
from each of the other two pieces. Then, knowing the weights of the three
pieces and the total weight of the cortex, the number of glomeruli in the
whole kidney was obtained.* The first dog weighed 11 kerm., its right kidney
weighed 34:5 grm.,and the total number of glomeruli was 142,000. A kidney
of a second dog, weighing a little over 8 krgm., contained 125,000 glomeruli.
Employing a different method, Petert+ calculated the number of glomeruli
in the dog’s kidney as 300,000. He does not give the weight of the kidney,
nor does the method he employed appear to me comparable in accuracy with
that above described. I have not been able to find any further record of
enumerations of the glomeruli in the dog’s kidney, and I wish to acknowledge
my great indebtedness to Miss M. G. Thackrah for carrying out this very
tedious piece of work.
Measurements of the lumina of the tubules in their several parts were
now made, as also approximate estimates of the lengths of the tubules based
upon the measurements of Peter.
The average results obtained from these measurements in the case of the
first kidney were :—
Length. Diameter.
cm. p.
Proximal convoluted tubule ...... ie 12
Loop of Henle—
Descendineslimibiee esses. 0-9 10
Aseendinorlimalai, cette sense. see 0-9 9
Distal convoluted tubule ......... 0:2 18
Collecimettulbulle sts. seeseer eens 2°2 16
* This is practically the method originally adopted by Huschke in 1828 (‘ Isis,’ vol. 21,
p- 550).
+ Peter, ‘Verhandl. D. Anat. Ges., Wiirzburg,’ 1907, p. 120.
A New Conception of the Glomerular Function. 577
The diuresis at the time the kidney pedicle was ligatured was 1 c.c. per
minute.
From the formula for the flow of liquids along narrow tubes
p = 8ln/7r* times flow in cubic centimetres per second dynes per square
centimetre,
where / is the length of the tubule in centimetres,
m is the coefficient of viscosity, and
7 is the radius of the tube in centimetres.
Taking 7 as 719 x 107, the coefficient of viscosity of water at 35° C., we
have
2 —5
— 8x7i19x10 1 2 1016 , 5 dynes per square centimetre,
1 "60 ° 142000 °
7 being now expressed in microns; or
eS 10) jm, Hg ease mg
Consequently, for a flow of 1 ¢.c. per minute,
p. mm. Hg.
p per centimetre of tubule, when 7 = 4°5, = 39°29,
Tee — EOS
(P(e ee
(job = Be,
T= 9, = 2°46.
Hence pressure-head required for—
mm. Hg.
Proximal convoluted tubule...... = 12x1243 = 14916
Loop of Henle—
Wescendimeplimaby kea-cep ees ees = 0°9 x 25°78 = 23°212
Ascending imabee tereceee eevee si-r = 0:9 x 39:29 = 35°361
Distal convoluted tubule ......... = 02x 246= 0-492
Collectmestubulessnes-t kee cis = 22x 393 = 8-646
Total pressure-head......... 82°627
In the case of the second kidney, with 125,000 tubules, the measurements
were :— Length. Diameter.
em. p.
Proximal convoluted tubule...... 1:0 12
Loop of Henle—
Wescendine lim br sees 0:8 10
Ascendinpilimiby.:s.ts.1--ceetes 0:8 10
Distal convoluted tubule ......... 0:2 18
Collecting tubule) 2ih...Net-... 2°0 8
_ And, with a diuresis of 0°85 c.c. per minute, the pressure-head required works
out to 74:1 mm. Hg.
578 Prof. T. G. Brodie.
I do not wish to lay too great a stress upon the actual pressure-head thus
obtained, for the possible errors in the measurements are many. It is, for
instance, impossible to obtain anything but an approximation to the lengths
of the successive portions of the tubule, and also the measurements of their
lumina can only be approximate, for they are undoubtedly altered during
fixation. Also I have supposed all the tubules to have equal lumina, and
have neglected to take into account those tubules which were at rest. To
obtain the total pressure within Bowman’s capsule a factor for the velocity
head should be added to the pressure-head already calculated, but it is so
small that we may omit it. (The mean velocity within the narrowest portion
of the tubule amounts to about 1 mm. per second.)
The important point is that during an active diuresis a pressure-head of
the order of 80 mm Hg. may be needed within Bowman’s capsule to drive the
fluid secreted there down the tubule.
The mean aortic blood-pressure in the first experiment was 120 mm. Hg,
and in the second 115 mm. If we allow 30-35 mm. Hg as the loss of
pressure-head between the aorta and the glomerular capillaries when the
afferent glomerular vessels are dilated, the blood-pressure within the capillary
loops would amount to 90-85 mm. Hg in the first experiment, and 85-80 mm.
in the second. Hence, on these figures, practically the whole of the blood
pressure-head is required to set up a pressure-head in the fluid within the
capsule sufficient to drive the secreted fluid down the tubule. Bearing in
mind that the estimates given are only approximate, I conclude that the
pressure-head within Bowman’s capsule only differs from the pressure-head
within the glomerular loops by the pressure required to stretch the walls of
the loops. This latter probably does not amount to more than one or two
millimetres of mercury.
If in the light of these arguments we criticise once more the assumptions
made by Ludwig’s theory, we see that that theory becomes less tenable than
ever. In the first place, when the kidney is secreting water at its fastest
rate, the pressure difference available for filtration is reduced to a minimum
At lower rates of secretion, of course, a pressure difference might be available.
In the second place, the assumption must be made that the volume of water
discharged from the glomerulus is from 30 to 70 times greater than the
volume of water entering the pelvis of the kidney. Hence a very much
greater pressure-head would be required to drive that fluid down the tubule,
though not 30 to 70 times greater than the pressure required to drive a
volume equal to that of the discharged urine, since the fluid has to be driven
only as far as the absorbing surface. But as the absorbing surface would
have to be taken as extending at least to the end of the ascending limb of
A New Conception of the Glomerular Function. 579
the loop of Henle, i.e. along considerably more than one-half of the whole
tubule, and the whole length of the narrowest part of the tubule, the pressure-
head required would be enormous, certainly many times greater than the
glomerular blood-pressure. We should, therefore, be compelled to ascribe
to the cells secreting the water the power of setting up a very high hydro-
static pressure, and all the evidence is strongly against any such view. A
pressure within Bowman’s capsule greater than the blood-pressure would at
once Jead to the closure of the glomerular loops and arrest of the circulation.
This is the main reason why neither the cells of Bowman’s capsule, nor these
covering the glomerular tufts, nor those of the convoluted tubule, possess the
power of setting up a hydrostatic pressure.
The quantity of energy imparted by the blood to the glomerular secretion
is only a small percentage of its total amount. Thusif V be the minute
volume of blood flowing through the glomerulus, and v the minute volume of
glomerular secretion, then V c.c. of blood enter the glomerular capsule, and
V—vc.e. leave it. If p be the pressure-head in the glomerular loops, the
pressure energy of the blood entering is Vp, and that of the blood leaving is
(V—v)p. The pressure energy communicated to the glomerular secretion is
vp, and the ratio of this to the total pressure energy of the blood as it enters
is v/V. In the dog’s kidney V may have any value from 200 to 600 c.c.,and
v from 1 to 2 ¢.c. at the height of a diuresis. Thus the pressure energy given
up by the blood lies somewhere between 1 and 0:16 per cent. of its total
pressure energy.
Mistological Evidence.
In the next instance the test applied was that of microscopical examination
of the kidney after varying degrees of activity. If during diuresis fluid is
being forced at a considerable pressure from Bowman’s capsule down the
tubule, evidences of the action of this pressure should be indicated by changes
both in the glomerulus and in the tubule. It is very remarkable that
throughout the literature the accounts of changesin the glomerulus following
activity are so scanty, and many authors state that no changes whatever are
to be found (¢.g. Lamy and Mayer). Mackenzie and I therefore examined a
number of kidneys excised after diuresis had been induced under various
conditions, and found that decided changes are produced in the glomerulus
and tubule. We further found abundant evidence proving that the tubules
have been subjected to a high internal pressure. The full details of these
changes are given in aseparate paper.* The general results are as follows :—
On comparing a resting kidney with one that has been thrown into activity
* Vide p. 593.
580 Prof. T. G. Brodie.
by the injection of any diuretic which causes a free flow of water, the
differences between both glomeruli and convoluted tubules are of the most
striking character. These differences are illustrated in figs. 1 and 2,
which show the changes in the cortex under a low magnification. The
important points are the following:—In an active kidney the glomeruli are
always separated from the capsules, and usually there is a considerable
accumulation of fiuid in this position. The capsule is always rounded,
whereas in the resting kidney the capsule lies in contact with the glomerulus,
and the whole structure is usually irregularly polyhedral in shape. In an
active kidney, in contradistinction to the resting, the individual loops of the
glomerulus are frequently separated from one another and stand out clearly.
The glomerulus also has a very characteristic vacuolated appearance, due, we
think, to dilated capillaries, from which the red blood corpuscles have in
some way or other been removed or destroyed, possibly post mortem.
When examining two such kidneys under a low power of magnification the
contrast is most striking. In the resting kidney the glomeruli are far from
conspicuous, and have to be sought for. In the active kidney, on the other
hand, they stand out at once as the most conspicuous objects in the field of
view.
The changes in the tubules are just as striking. Whereas a resting proximal
convoluted tubule possesses no lumen, one in activity has a large lumen. This
is true both of the proximal and the distal tubules. Moreover, in the resting
kidney the tubules are very much twisted on themselves and form very
complicated foldings, whilst in the active kidney the appearances indicate
that the tubule is as far as possible straightened out. All these several points
prove quite clearly that the tubules have been subjected to some high fluid
pressure from within.
The changes accompanying activity are strikingly emphasised when we
measure the diameters of these several structures. In the case of the
glomeruli and capsules, in addition to measurements in diameters at right
angles to one another, approximate calculations of their volumes were also
made.
In one experiment which we may take as typical we obtained the following
results :—
Resting. After activity.
Volume of capsule............ 2. 83* 220
i glomerulus® inci... 0: 80 ll
3 fluid in capsule ...... 3 109
* These figures can be converted into cubic millimetres by multiplying them
by 4:2 x 10-8,
A New Conception of the Glomerular Function. 581
The differences are therefore very great. The capacity of Bowman’s
capsule in the active kidney is nearly three times that of the capsule in the
resting kidney, chiefly on account of the big accumulation of fluid within the
capsule.
The volume of the glomerulus has also increased, though only by 40 per
cent. Such measurements prove, therefore, that both the glomerulus and the
capsule of Bowman are extensible structures, and that a considerable volume
_ of fluid accumulates in the capsule during activity.
In drawing deductions from these measurements, full attention must be
paid to possible alterations occurring after the kidney is excised. To obviate
change as far as possible in these experiments, the artery, vein and ureter
were ligatured close to the hilum at the instant the experiment was to be
stopped, using a single coarse ligature. The kidney was then excised, rapidly
weighed, and placed at once in the formalin fixative. If active diuresis were
in progress, the kidney at the moment of ligature was hard and tense, but
within a few seconds after application of the ligature became quite soft, chiefly
on account of escape of blood through the Capsule. We found it impossible
toavoid this. The question therefore arises: Does this fall of tension within
the kidney substance involve a change in distribution of the fluid contained
within the tubule and capsule? It is possible, for instance, that fluid is forced
back from the distended tubule into the capsule. Possibly this may be the
cause of some of the increase in volume of the capsule seen in our experiments,
but the changes are too great to be wholly, or even largely, explicable in this
way. There is yet another post-mortem change we think possible, viz., that
before the fixative has time to penetrate and reach the glomeruli, the cells
forming the loops die and permit osmotic effects to take place through them
between the fluid in the capsule and the blood. Fluid would pass into the
blood, and we think it possible that this fluid is so low in salinity as to lake
some of the corpuscles, thus producing the vacuolated appearance described
above.
In the same experiment the measurements of the diameters of the proximal
and distal convoluted tubules and of their lumina were as follows :—
Resting. Active.
B. EB.
Proximal convoluted tubule—
Transverse diameter ............ 44-0 43:0
Lumen, diameter ............... 0:0 19°4
Distal convoluted tubule—
Transverse diameter ............ 25°4 318
Lumen, diameter...........000000. 11:0 21°8
582 Prof. T. G. Brodie.
_ This is fairly typical of the results obtained in all our experiments. We
found it to be practically a universal rule that the external diameter of the
proximal convoluted tubule remained unaltered, or showed but a slight
increase or decrease. The marked change during activity is the production
of a big lumen within the proximal tubule. The idea given by an examination
of the sections is that the loops of the convoluted tubule have been opened
out and stretched in length. They are in nearly all instances circular in
outline, and invariably, as just stated, there isa very wide lumen. The distal
convoluted tubule in contradistinction is nearly always increased in diameter
in the active state, and the lumen greatly increased, often doubled, although
this tubule has invariably a rather large lumen even in the resting kidney.
We have not yet carried out a sufficient number of measurements of the
remaining portions of the tubule to warrant us making any decided statement
as to the changes they undergo. It is clear that the limbs of the loop of
Henle are both distended, and often the collecting tubules show very distinct
expansion.
The next modification in our experiments consisted in comparing the two
kidneys after active diuresis, one kidney having been previously stripped of
its Capsule.
The kidney is very characteristically enclosed in a strong and
practically inextensible Capsule*, and my view of the meaning of
the glomerulus offers an explanation of that fact. As fluid is
secreted into Bowman’s capsule by the epithelium covering the
glomerular loops, and possibly also by the epithelium of the capsule,
the blood-pressure acting within the glomerular loops is transmitted directly
to that fluid and through it to the wall of Bowman’s capsule. This latter,
as we have seen, is extensible and might be ruptured if the distension
were carried too far. Again, fluid is at once forced into the convoluted
tubule, and that also might be ruptured if overdistended. To prevent any
dangerous overdistension the whole of the structures are enclosed in a firm
Capsule. That this distension does take place on activity is amply proved in
a variety of ways. Firstly, as shown above, the histological appearances
demonstrate it. Secondly, if in an experiment we excise one kidney at the
commencement, then excite diuresis, and at its height ligature the pedicle of
the other kidney to prevent escape of urine from the tubules, and we then
weigh the two kidneys, the latter often shows an increase in weight amount-
ing to about 30 per cent. This increase in weight is not due to blood, for
on excision the blood escapes more readily from such a kidney than from a
* In order to avoid confusion between the Capsule of the kidney and Bowman’s
capsule, I will when referring to the former distinguish it by a capital.
A New Conception of the Glomerular Function. 583
resting kidney. In the third place I have often observed the following
changes during the course of an oncometric experiment, viz., a large
increase in the volume of the kidney, a free flow of urine, but a decrease in
the rate of blood flow through the kidney. Here the plethysmographic
increase is due to an accumulation of urine within the capsules and tubules.
Lastly, if we examine a kidney at the height of a diuresis we always find it
very hard and tense. The Capsule is distended to its fullest degree. If we
attempt to make such a kidney expand still further by temporarily clamping
the vein we fail completely. We see then that some of the tension set up
by the blood-pressure in the glomeruli is transmitted through the capsule
wall and the walls of the tubules to the general renal tissues. How much
pressure is thus transmitted depends upon the resistance to distension offered
by Bowman’s capsule and the walls of the convoluted tubules. Their
structure, particularly that of the capsule, indicates that they probably offer
a fairly considerable resistance. We could get an estimate of this by finding
the difference between the blood-pressure in the glomeruli and the general
tension of the kidney substance within its Capsule. I made some attempts
to measure this latter during active diuresis, but at present have not obtained
any very accurate results. As far as they go they indicate a tension of about
40 mm. Hg.
If this be the true meaning of the kidney Capsule then, if we remove it
before exciting diuresis, the kidney ought to expand still further as compared
to the intact one, and the amount of that further expansion should depend
upon the general rigidity of the kidney substance and the amount of con-
nective tissue it contains. Our experiments proved this to be the case. The
weight of such a kidney compared to one with the Capsule untouched was
always greater, especially in the rabbit’s kidney.. In the cat there are
a number of incomplete septa running transversely towards the
hilum, and on active diuresis the kidney substance bulges notably between
these, giving the appearance of constricted grooves in the bottom of which
veins run. This relatively greater increase in volume of the kidney as a
whole is also found in the several parts of the tubule, and when we measured
the tubules and glomeruli in such kidneys, the differences were very distinct.
For instance, in one experiment the right kidney was untouched, and the left
decapsulated. The following approximate volumes of the capsule and
glomerulus after diuresis were obtained :—
R. L.
Volume of capsule ............... 205 257
# PIOMETUS * 64 faciganan 3s 128 151
i lip yates ay We 27 106
| 584 | Prof. T. G. Brodie.
The diameter of the tubules was as follows :—
Proximal convoluted tubule— “ .
External diameters sisted sedaes 44:8 480
Me nee wees eh eee an ee ea 13:0 19°8
Distal convoluted tubule—
External diameter ...............005 33:2 39-2
Taunane tas S555. ead ee ie 248 ~ 29:2
The expansion then is found in all parts, and is obviously brought about
by a distending force acting within the tubules.
Yet another means of testing the theory which presented itself was to
observe the effect of obstructing the exit of urine down the ureter. In the
first set of experiments a diuresis was set up, and at its height the ureter on
one side was suddenly clamped. Five to fifteen minutes later the two kidneys
were exposed, their condition noted, and then the pedicles ligatured as close
as possible to the hilum. The kidneys were then removed and weighed. As
was to be expected, a kidney obstructed in this manner is very distended and
tense within its Capsule. The weights found in one experiment in which the
right kidney was obstructed at the height of diuresis, and the left secreting
freely, were as follows :—
grm
Weight of R. kidney ......... 15°5
F, eM cdneyaeeeeeece ABw
The right kidney was very tense, appeared almost bloodless, and was
distinctly lobulated. The left kidney was distinctly softer than the right
and also more vascular. The approximate volumes of the capsules and
glomeruli were :—
L. R.
Volume of capsule ............ 86 146
. glomerulus ...... 69 89
= Fliit dfontpberco ti tate os iy 57
The measurements of the tubules were :—
Be H
Proximal convoluted tubule—
iRxternalidiameber seeseessceeeee 39°2 39°4
hm en eee ee a sree 6°6 14:0
Distal convoluted tubule——
External diameter ............... 22°8 28:2
TEAM Me ieee ee 12°6 18:4
A New Conception of the Glomerular Function. 585
Lastly, in an experiment in which an obstructed kidney was compared to
a decapsulated one we found that the former procedure produced more effect
than decapsulation.
Maximum Ureter Pressure—Another series of observations which receive
a satisfactory explanation is that in which the maximum ureter pressure is
measured. According to my theory, fluid should be forced out of the tubules
only when the pressure within the ureter lies below the maximum glomerular
blood-pressure. This of course assumes that the tubular epithelium in
secreting does not set up any appreciable hydrostatic pressure. From this
point of view the measurement of the maximum ureter pressure should be a
means of determining the intraglomerular blood-pressure, always supposing
that none of that pressure is taken up by the walls of the glomerular loops.
Now the measurements of the maximum ureter pressure fit in perfectly with
this conception. In an animal whose aortic blood-pressure is about 120 mm.
Hg, the maximum ureter pressure is usually found to be somewhere between
80 mm. and 90 mm. Hg, that is, a loss of pressure-head of some 30 to 40
mm. Hg occurs between the aorta and the glomerular capillaries. This is
distinctly less than is the case for most systemic vessels, and fits in well with
our knowledge of the relatively wide and short path of the blood stream from
the aorta to the glomerulus. We have only to recall how fast the blood may
flow through the kidney to realise that the glomerular capillary pressure
during activity must stand at a greater height than the ordinary systemic
capillary pressure.
Let us then return to a general restatement of the whole problem. I
have given evidence that the glomerulus, Bowman’s capsule and certain parts
of the tubules are elastic structures, and that their overdistension is prevented
by the general inextensibility of the connective tissue framework and of the
Capsule. Consequently as soon as fluid is secreted by the glomerular surface
into the capsule, the glomerular capillary pressure comes into play, and some
part of that pressure is transmitted through Bowman’s capsule to the tubules
immediately outside. Then as the secretion continues to accumulate, the
kidney expands to fill the Capsule, and the pressure within the Capsule
reaches its maximum. Hence we may regard the glomeruli as a number of
expanding vascular tufts, lying within a space which cannot expand beyond
a certain point, consequently the expansion of the glomeruli expels any fluid
free to move outwards. It is as if we were dealing with a sponge work filled
with fluid, and enclosed in a capsule which it completely fills. Distributed
through the sponge are a number of elastic structures which can be expanded
by a fluid pressure acting from within, their expansion necessarily compressing
the sponge, @.e, expelling the fluid from between its interstices. This analogy
586 ; prot @ Brodie.
is of course incomplete, in that it takes no account of the tubular structure
and the facts that the pressure is set up in the fluid within the tubules and
that the walls of the tubules offer some resistance to expansion. The first
effects of the glomerular pressure will therefore be to distend the capsule
and the first convoluted tubule, 7.c. to increase its lumen, thus offering less
resistance to the flow of fluid along the tubule. In this distension the pulsa-
tion of the glomerular vessels is probably utilised. Also the more rapid the
flow along the tubule the greater the pressure gradient, and the smaller
the pressure transmitted through the walls of the tubules to the
general kidney substance. We must therefore expect to find a distinct
difference between the intratubular pressure and the intra-Capsular pressure,
and while fluid is moving down the tubule the two could only be equal at
the point where the tubule leaves that part of the kidney substance where .
the pressure is raised. This region is limited as we shall see by the branching
arches of the renal vessels in the intermediate zone.
There is yet another feature of the renal structure and form which is
capable of interpretation by this theory. This is the general shape of the
mammalian kidney, so typical as to give its name to all structures in any
way resembling it. The kidney is very typically constructed of a cortical
mass enveloping a medullary portion. The blood-vessels form a set of arches
between these two parts. My suggestion is that this arched system of
vessels forms a more or less rigid base upon which the cortex lies. Conse-
quently when, in activity, the pressure in the general renal tissue rises
through the activity of the glomeruli it is restricted in the first instance to
the cortex. The cortex, so to speak, becomes compressed between the rigid
Capsule and the firmly distended arterial arches. From this general pressure
the medullary portion is relieved, and it is a most significant fact that the
loops of Henle lie within this region, where there is probably but little
external pressure. Apparently, then, the difference in state between the
tubules in the cortex and those in the medulla is that there is a high
pressure on both internal and external surfaces of the tubules lying in the
cortex, whereas in the medulla the pressure may be acting chiefly, possibly
entirely, from the inner surface of the loops only. In this connection I have
frequently observed the following most notable result :—If at the height of
a diuresis whilst urine is flowing freely the ureter be ligatured, and after
about 20 minutes the pedicle be tied off and the kidney removed, it will
be found that the pelvis is widely distended with fluid, and usually the
pyramid is compressed towards the cortex until it forms an almost
insignificant structure projecting into the cavity of the pelvis. Histo-
A New Conception of the Glomerular Function. 587
logically the tubules within such a collapsed pyramid are observed to be
flattened and empty.
It is possible that some or even all of this compression might be post
mortem, but I think that it is ante mortem, since it 1s only found if sufficient
time be allowed to lapse between the ligaturing of the ureter and the removal
of the kidney. The longer the interval the more marked is the compression.
I think the compression is produced in the following way :—After the ureter
has been ligatured urine continues for a time to be expelled into the pelvis,
and gradually the pressure there rises. Fluid will continue to be forced
into the pelvis in gradually decreasing volume until the pressure reaches
that of the glomerular capillary blood-pressure. The further distension of
the pelvis and compression of the medulla is probably produced through the
pulsatory variations of pressure in the cortex. The systolic pressure, by
the expansion of the glomeruli and arteries, suddenly raises the tension
throughout the whole cortex; this expels a little of the fluid from the
terminal portions of the tubules into the pelvis, whose pressure then becomes
greater than diastolic pressure. As the pressure falls in diastole a point is
reached at which the cortical pressure is below the pressure in the pelvis,
that is below the pressure in the fluid contained within the loops of Henle
and the collecting tubules. Accordingly these latter are emptied or partially
emptied into the cortical tubules, while the lower ends of the collecting
tubules are compressed and act as valves, preventing any return flow from
the pelvis up the tubule. In this way more and more fluid is gradually
collected within the pelvis at the expense of the medulla.
If, as I think is the case, we may divide the kidney substance into two
parts, in one of which the whole tubule is exposed to a considerable pressure,
both internal and external, while in the other region the pressure is largely
within the tubule, the difference must have some important physiological
meaning. It is most significant that the loops of Henle are carried down
into this region of low external pressure, In different animals the loops of
Henle show many diversities of form, more particularly in length, and it is
certainly a striking fact that in some animals the major number of loops are
short, and either lie completely within the cortex or only descend into the
outermost portions of the medulla. It has been pointed out that the animals
with very short loops are those which secrete a dilute urine, whilst those
in which the loop penetrates far into the medulla secrete a concentrated
urine. Hence it may be that this loop effects a certain amount of absorp-
tion, a function which would be aided by a pressure difference acting from
within the tubule.
To test my theory further, and in the hope of gaining some evidence of the
VOL. LXXXVII.—B. a Sf
588 Prof. T. G. Brodie.
respective activities of the different parts of the renal apparatus, another
series of experiments was performed, in which the action of diuretics upon
animals whose blood-pressure had been lowered by section of the spinal cord
was tested. It was necessary to employ rabbits for these observations, since
in both the cat and the dog the blood-pressure remains high enough after
section of the cord to enable the kidney to secrete quite freely when a
diuretic is administered. In the rabbit the blood-pressure falls to about
30 mm. Hg, and even though we injected large doses of saline and other
diuretics we never obtained a single drop of urine from the kidneys. The
plan of experiment therefore was to excise one kidney some 10 to 20 minutes
after division of the spinal cord, then inject the diuretic to be studied, and
half-an-hour later to remove the other kidney. In this way evidence was
obtained indicating the point of action of various diuretics. Without going
into the results in detail, I may state that the glomerulus is excited to secrete
by most of the diuretics of the saline group. Thus activity was well marked
after sodium sulphate, urea, or dextrose; it was excited also by caffeine, but
completely absent after phloridzin. In the tubules the results were equally
striking, especially in the case of phloridzin, and in a minor degree in the
case of caffeine. In no instance was a large lumen produced, and the external
diameters of the convoluted tubules were only slightly increased. The contents
of the lumen consisted of fairly large secretion droplets, the droplets being
enclosed in membranes which stained with Weigert’s hematoxylin, and fairly
well with eosin, These results were chiefly observed in the proximal con-
voluted tubule. With the low blood-pressure there was never the slightest
indication of any marked distension of the tubule in any part of its course.
The glomeruli were never found secreting very actively, but were always
found separated from the capsular epithelium by a distinct though small
accumulation of fluid.
An examination of the embryology of the renal tubule bears out the views
I have expressed. Originally, the excreting apparatus was a long tubule
opening at one end into the body cavity, and at the other on to the surface.
This tubule was lined throughout by a ciliated epithelium, which provided
the necessary motor mechanism for the expulsion of the secretion. Later, the
glomerulus was developed from the dorsal wall of the body cavity and received
a large and important blood supply from the aorta. Possibly its original
function was to secrete a watery fluid into the body cavity, and this in some
way served the renal tubule. The arrangement of its vessels as large loops
projecting from the ccelomic wall, even at this early stage, tends to indicate
that it was employed as a means of raising the fluid pressure within the
A New Conception of the Glomerular Function. 589
celom. In the next stage of development that part of the body cavity which
contained the orifices of the renal tubules and the glomeruli became largely
constricted off from the rest, and by means of imperfect septa the glomeruli
also became partially separated from one another. This indicates that the
function of the glomerulus has now been restricted almost solely to work in
association with renal excretion. Later, this becomes entirely the case by the
complete separation of that portion of the ccelom from the rest. Each
glomerulus then works in conjunction with a renal tubule, but at first the
number of the latter is largely in excess of the former. The material
secreted at the glomerular surface is now conducted entirely to the tubule, as
is also any formed by the isolated portion of the ccelomic endothelium. It
is very significant that as soon as the relationship between glomerulus and
tubule is completed the latter loses its cilia, only the cells of the neck of the
tubule retaining them in some animals. This indicates that some other
mechanism for the propulsion of fluid down the tubule has taken the place of
the ciliary movement. This, according to my view, is the propulsive action
of the glomerular capillary loops.
Previous Work Bearing upon the Subject.
L. Hill, in discussing the general distribution of pressure through a soft
and yielding animal tissue, arrives at the conclusion that filtration is an
impossible mechanism at the glomerular surface. With much that Hill
expresses in his paper on “Filtration in the Living Organism,’* I am in
complete agreement, but in several points I think he is incorrrect. Thus,
he considers that the glomerular capillary pressure must be transmitted in
undiminished amount throughout the whole renal tissue. This implies that
the wall of Bowman’s capsule is incapable of offering any resistance to
extension, and similarly, too, for the walls of the tubule. Our measurements
show, however, that while these structures expand, they offer resistance to
expansion. They indicate that a higher pressure has been acting on the
internal surface of the tubule than on the outer, and especially until a
sufficient dilatation has been produced to make the kidney substance as
a whole expand, and thus render the Capsule tense. From that point on,
the tension in the kidney substance rapidly rises. I have found by measure-
ments of the blood flow that at this point the blood flow falls, due, that is, to
compression of the capillaries around the convoluted tubules and of the
renal veins. The fact that the capillary system which originates this
pressure consists of characteristic tufts which lie entirely within capsules
is very significant. In certain forms of tubular nephritis, in which the
* © Biochem. Journ.,’ 1906, vol. 1, p. 55.
Ned
590 . Prof. T. G. Brodie.
tubules are blocked or obliterated, and have been so for a considerable time,
the capsules are often found distended to a volume even ten times greater
than the normal volume. In these cases the glomerulus is collapsed and
shrunken to a minute structure, which appearsas a mere projection into the
swollen capsules.
In my opinion, too, Hill does not allow a sufficient fall in pressure-head
between the glomerular capillaries and the tubule capillaries. The efferent
blood-vessel of the glomerulus is of small diameter and fairly long. Hence
with the exceedingly rapid blood flow observed during diuresis, there must of
necessity be a considerable pressure difference between these two capillary
systems. I cannot, therefore, agree with Hill’s statement: “The pressure of
the secretion cannot be normally greater than the pressure in the veins, for
otherwise the secretory pressure would compress the veins”; nor, again,
with the statement: “The secretion moves onward, I take it, by phenomena
of adsorption.”
At about the same time Filehne and Biberfeld* reasoned that filtration at
the glomerular surface was an impossibility, since there were no firm support-
ing structures capable of resisting any pressure. They, too, consider that the
glomerular capillary pressure is at once transmitted through the whole renal
substance, leaving no pressure difference available for filtration through the
glomerular surface. While agreeing with them that but a very minute
pressure difference can exist between the glomerular blood-pressure and the
pressure of the secreted fluid within Bowman’s capsule, I am in disaccordance
with them, for reasons already stated, in their idea that the glomerular
pressure is at once transmitted in undiminished amount to the general renal
substance.
Shortly after I had expressed my views as to the work of the glomerulus,
Lamy and Mayert published a paper in which they suggested that the
glomerulus by its pulsation acted as a kind of heart, and by its piston-like
movements drove the liquid forward in the tubule, and favoured its discharge
by overcoming the friction and the capillarity of the tubule. They do not
consider that the glomerulus plays any important part in the secretion of
water. If it secretes any at all, this is in their opinion quite a minor
role. According to them the glomerulus performs mechanical work solely
by virtue of its pulsation, and consequently their view differs widely from
mine. Iam, in the first place, in wide disagreement with them in that I
consider that the main bulk of the water is secreted by the glomerular surface.
There is abundant evidence to prove this. I need only refer to the work
* © Pfliiger’s Archiv,’ 1906, vol. 111, p. 1.
+ ‘Journ. de Physiol.,’ 1906, vol. 7, p. 660.
A New Conception of the Glomerular Function. 591
of Miss Cullis upon secretion in the frog’s kidney,* or to the results I have
briefly described above upon secretion in the rabbit’s kidney after division of
the spinal cord. As is seen from what I have stated, the fact that the
glomerulus pulsates has but little bearing, if any, upon its work in propelling
the secreted water along the tubule. That pulsation is unimportant in the
propulsor action of the glomerulus is borne out by the fact that the urine
flows quite freely along the ureter of an excised kidney perfused with fluid
at constant pressure, and if in these cases the perfusing fluid be of correct
composition, the kidney presents at the end of the experiment appearances
exactly comparable to those found by Mackenzie and myself after active
diuresis in the intact animal. It is possible that pulsation may play a part
in producing the primary dilatation of the convoluted tubule. In an artificial
schema representing the glomerulus and tubule, I have found that the volume
of fluid driven along the capillary tube by a pressure made to vary in imitation
of the pulse variations is exactly the same as if a steady pressure at the mean
height of the varying one is used. This indeed was to be expected from
theoretical reasons. The value of a varying pressure only arises when the
tubule along which the fluid is to be driven has first of all to be expanded.
In conelusion, then, we may summarise what I have said in the following
way :—
The glomerulus is a secreting surface whose chief function is to secrete the
main bulk of the water of the urine, but it is also thrown into activity by
such substances as salts, urea, dextrose and caffeine. Its highly characteristic
shape is to enable it to act as a means of setting up a pressure-head sufficient
in amount to drive the secreted water down the long urinary tubule. The
pressure originating from this is also transmitted in some degree through
Bowman’s capsule to the general tissues of the cortex, thereby exerting a
pressure upon the external surfaces of all the tubules lying in the cortex.
To what degree the pressure on the external surfaces of the convoluted and
other tubules lies below the glomerular capillary pressure I am not yet able
to state definitely. The fact that the convoluted tubules show such marked
evidences of having been subjected to a high internal pressure certainly
indicates a considerable diminution. I have also given reasons for believing
that the general pressure conditions so typical of the cortex are non-existent
in the medulla; there, apparently, the internal pressure acts upon the loops
of Henle in undiminished amount, and must be supported either by the
basement membrane of those tubules, or by the general tissue of the medulla
itself. At present the former seems the more probable. Lastly I have given
evidence attained by the application of yet another method, which enables us
* ‘Journ. of Physiol.,’ 1906, vol. 34, p. 250.
592 A New Conception of the Glomerular Function.
to determine from histological evidence the part of the urinary apparatus
thrown into activity by the different urine exciting substances.
[Addendum.—Shortly after I delivered this lecture before the Royal Society, letters
appeared in the ‘ Lancet’ and the ‘ British Medical Journal’ by Mr. Wm. Woods Smyth,
claiming that his brother, Dr. A. W. Smyth, had over 30 years ago anticipated the views
I now expressed. Dr. Smyth’s views of the function of the kidney appeared in a
pamphlet by Mr. John Gamgee, in the ‘ New Orleans Medical and Surgical Journal’ for
May, 1880, and were based upon microscopic examination of the kidney, and upon the
fact that the kidney pulsated with each heart-beat. As far as I am aware, no reference
to his views has ever appeared in the literature upon the kidney. They concerned the
glomerulus and the circulation through the kidney. He denies the existence of any
“ connection between the capsule of the Malpighian body and the interior of a uriniferous
tubule,” and also “having observed that the hyaline membrane, enclosing each glomerule,
was unprovided with epithelium, essential to every secreting structure, Dr. Smyth
perceived that so delicate a sac would rupture, and the plexus be destroyed, if subjected
to hydrostatic pressure, either during secretion or from accidental regurgitation.” But
the main point in relation to this lecture is his view of the mode of working of the
glomerulus. This he describes in the following terms :—“ Every heart-beat is attended
by turgescence of the glomerule. The loops, from their position and form, must swell
outward and inward in all directions, and, constricting the efferent vessel, momentarily
impede the blood’s exit. At each cardiac diastole, the arterial column sustaining the
blood in its channel, the Malpighian loops recoil and fill the current in the secreting
vascular rete. And this is Dr. Smyth’s view of the special function of the Malpighian
bodies. Their alternate turgescence constituting a ‘rhythmic vascular impulse,’ a
uniform, safe, and sufficient expelling pressure is maintained on the coiled tubes, and,
indeed, on the whole excreting structure of the kidney. Those acquainted with the
laws which govern the flow of liquids can readily understand that the power required
to maintain a circulation, beyond the coils of the glomerule, would be destroyed, if a
mere physical transudation could occur through the loops, so well disposed to bring the
very active pulsation to bear on the maintenance of a circulation.”
“The unmistakable constriction of the efferent vessel, on the filling of each glomerule,
causes an alternation between clearance of the tubuli and the flow of blood in the
secreting vascular rete. The glomerules are filled during the heart’s systole; the
secreting rete is turgid during the heart’s diastole.”
Undoubtedly Dr. Smyth’s conjecture was in the right direction, but his erroneous
conclusion that Bowman's capsule did not open into the tubule, and the fact that he
ascribed all the expelling power of the glomerulus to its pulsation, will indicate
sufficiently the great divergence of his views from those I have expressed in my lecture. ]
DESCRIPTION OF PLATE.
Fig. 1.—Microphotograph of Cortex of Kidney of Cat, after period of rest, showing
absence of lumen in convoluted tubules and irregular outline of glomeruli.
x 120.
Fig. 2.—Microphotograph of Cortex of Kidney of Cat, after sulphate diuresis, showing
widely dilated tubules and distended capsules, which are now rounded and
contain much fluid. The glomeruli are larger than in the resting kidney, but
not filling the capsules. x 120.
Brodie. Roy. Soc. Proc., B, vol. 87, Plate 26.
593
On Changes in the Glomeruli and Tubules of the Kidney
accompanying Activity.
By T. G. Bropiz, M.D., F.R.S., and J. J. MAckENziE, M.B.
(Received December 9, 1912,—Read February 20, 1913.)
(From the Physiological and Pathological Laboratories of the University of Toronto.)
[ Puate 27.]
The experiments described in this paper were designed to test the
correctness of the view put forward by one of us,* namely, that the
glomerulus is a propulsor. If this view be correct, the marked dilatation
of the tubules, which is so prominent a feature in a kidney after active
diuresis, is simply the expression of the forcible distension of the tubule
from within, effected by the discharge of fluid from the glomerulus down
the tubule, the active propelling and dilating force being the intraglomerular
blood-pressure transmitted through the glomerular capillary cells and
epithelium. As, however, the condition of the glomerulus after active
secretion has not been made the subject of extensive observation, it seemed
probable that a thorough study of the alterations in size and appearance
of both tubule and glomerulus might give many points of importance in
criticising the propulsion theory. Thus, if the capsule be free to expand, we
may find it enlarged after active diuresis; and again, if the propulsive action
of the glomerulus is complete and instantaneous, we should find the
glomerulus filling Bowman’s capsule completely under all conditions. But
it was also possible that, after a very free secretion of water, there might be
a considerable accumulation of fluid between the glomerulus and the capsule
wall. We therefore measured the sizes of the capsules, the glomeruli and the
tubules in kidneys, before and after diuresis had been set up under varying
conditions. The more important of these states were :—
1. The kidney at rest.
2. The kidney secreting freely. This we term an “active free” kidney.
3. Decapsulated and secreting freely. This we term an “active decapsu-
lated” kidney. The aim of the procedure was to test the explanation
offered by the theory as to the meaning of the Capsule.t
* Vide Croonian Lecture, supra.
+ As in the course of this paper we shall be referring constantly to the Capsule of the
kidney and to Bowman’s capsule, we will, in order to avoid needless repetition, distinguish
between them by employing a capital letter whenever we refer to the former.
594 Prof. Brodie and Mr. Mackenzie. On Changes in the
4, With the ureter ligatured. This we term an “active obstructed”
kidney.
We soon found that the different parts of the renal tubule, and more
especially of Bowman’s capsule and the glomerulus, varied considerably in
size in different animals, so that it is necessary in making comparisons to
use only, in the first instance, opposite kidneys in the same animal. Hence,
our series of experiments comprises each possible combination in the above-
named types of experiments.
All our experiments were performed upon cats anzsthetised with a
mixture of chloroform and ether.
In all experiments, the kidney was removed and fixed in the following way.
It was first carefully freed from subperitoneal fat, and a ligature then tied
tightly around the pedicle close to the hilum. A second ligature was next
tied around the pedicle a little nearer the aorta, and the pedicle divided
between the ligatures. The object of ligaturing the pedicle was to keep the
urine within the tubules, and as far as possible in the position it occupied
at the instant of ligature. The kidney was dropped intact into a beaker of
20-per-cent. formalin made up with 0°9 per cent. NaCl. The beaker and
solution had previously been weighed, and it was now weighed a second
time, giving the weight of the kidney. At the end of an hour, the kidney
was sliced into thin sections, fixation in formalin completed, and the pieces
imbedded and sections prepared. The following measurements were then
taken :—
1. An equatorial diameter of the capsule at right angles to the polar
diameter.
2. The polar diameter, z.c. one passing through the point of entrance of
the blood-vessels.
3. The greatest distance between the glomerulus and the capsule if the
two were not in contact.
4. The maximal diameter of a typical proximal convoluted tubule.
5. The diameter of its lumen. |
6and 7. Similar measurements of a typical section of the distal convoluted
tubule.
The glomeruli measured were taken at random, care being exercised only
to measure those in which the section passed centrally. This was generally
fairly easy to attain by taking those which showed the point of entrance of
the blood-vessel into the glomerulus. From these measurements, calculations
were made of the approximate volumes of the capsule and the glomerulus
respectively. To obtain these, we regarded the capsule as equal in volume
Glomerula and Tubules of the Kidney accompanying Activity. 595
to a sphere whose diameter was the mean of the two diameters of the capsule.
The figures representing volumes given in this paper were obtained by cubing
the mean radius of the capsule expressed in microns, and dividing it by 1000.
Hence, to convert the figures into cubic millimetres, they must be multiplied
by 42x107% The glomerulus was also compared to a sphere, whose
diameter was the diameter of the capsule minus the maximum space between
the glomerular surface and the capsular surface. The difference between the
two volumes thus ascertained gives us an approximate estimate of the
volume of the fluid contained within the capsule.
In measuring the tubules a section of a proximal convoluted tubule lying
near to the glomerulus was selected, and that section of the distal convoluted
tubule which hes close to the point of entrance of the vessels into the
glomerulus. Hence the proximal tubule probably belonged to the glomerulus
measured, and the distal tubule certainly did so belong.
I. Comparison between a Resting and an Active Kidney.
A. The Glomerulus and Capsule—There are always marked differences
between a resting and an active glomerulus. A resting glomerulus appears
to be made up of a dense tissue closely packed with nuclei (fig. 1). The
glomerular surface always lies in contact with the capsule wall, and the
whole structure is usually irregularly quadrangular in outline. After
activity the glomerulus stands away clearly from the capsule. The outline
of the glomerulus is lobular, and in structure it is much looser than the
resting glomerulus (fig. 2). It also appears to be filled with large vacuole-
like spaces approximately circular in section. The nuclei are well separated.
As a rule the number of blood corpuscles contained in the glomerular vessels
is quite small, far fewer than in the resting glomerulus. This we think
may be due to the expulsion of the blood from the capillary loops after
excision of the kidney, or to post-mortem laking of the corpuscles. The
latter may be produced by the diffusion of water from the capsule through
the walls of the capillary loops after the epithelial cells have died, and
before the fixative has had time to act upon them. This would account for
the very characteristic vacuolated appearance of the glomeruli already
alluded to.
We were never able to keep the blood in a kidney that was excised at the
height of activity. At the instant of excision such a kidney is hard and
tense, and instantly becomes soft when the first ligature is tied round the
pedicle. This is even the case though the vein be first ligatured, and though
the kidney may have been separated from its surrounding’ tissues before the
diuretic was administered in order to give ample time for closure of the
596 Prof. Brodie and Mr. Mackenzie. On Changes in the’
many small vessels passing through the Capsule. Even then there is a
distinct escape of blood through the Capsule, and the cortex rapidly pales
in colour as the tension falls. The greater the tension at the instant of
ligature, the greater is this paling of the cortex, and the sections of such
kidneys may show but traces of blood in any of the capillaries, and but little
in the veins.
The change in the shape of Bowman’s capsule when the kidney becomes
active is very distinctive. It becomes circular or elliptic in section, and
there is always fluid between the glomerulus and the capsule wall. In
many instances we have noted one other highly suggestive appearance.
This is that the first portion of the proximal tubule has, in cases in which
a free diuresis was established, been distended so as to appear almost a part
of the capsule wall. An instance of this is illustrated in fig. 3. It is a very
clear indication that the capsule and the first part of the convoluted tubule
have been subjected to a high internal pressure. There are further indica-
tions, moreover, that the capsule has been distended to a size much larger
than it appears in the section after fixation. The action of a high intra-
capsular pressure also adequately explains the change of shape from irregularly
quadrangular to spheroidal or ellipsoidal.
B. Lhe Tubules—The contrast between the tubules at rest and after they
have been in activity is just as striking, and in some particulars has already
been described by several observers. In this paper we deal entirely with
changes in the total diameter and in the lumen of the tubules, and, moreover,
restrict our attention for the most part to the two convoluted tubules.
The magnitude of these several changes is brought out by the following
measurements taken from Experiment 10. The measurements are in microns,
and each is the mean of 10 measurements :—
Expt. 10.—R. kidney resting. LL. kidney free.
R. L.
p. pe.
Glomeruli and capsules—
Equatorial diameter ............... 108-4 144°0
Polaridiameteriy -fastnaccescss oer 78:4 103°6
SPace: Sip saoswcsceeaasect emer renaectes 3°0 23'8
Hence
Mean diameter capsule ............ 93°4 123°8
- if glomerulus ...... 90:4 100:0
Approximate volume capsule ... 102 237
3 » glomerulus 92 125
” preup atin eke 10 112
Glomeruli and Tubules of the Kidney accompanying Activity. 597
Convoluted tubules—
Proximal. External diameter ... 41-4 41-4
Deanery ee eta kas 0-0 17-6
Distal. External diameter ... 21°2 32°4
Gia 0102) eS Pen catrancichs 2, 20°6
grm.
Weight of R. kidney......... 10°9
55 Sis @ yao. a. 16:2
These figures show most clearly how extensive a change in size of the
different parts of the renal tubule occurs when it is thrown into activity.
Thus the capacity of the capsule is more than doubled (to 232 per cent.),
chiefly because of the very large accumulation of fluid which has been
secreted. The glomerulus is, however, increased to 136 per cent. of the
volume of the glomerulus at rest. The differences are in reality still more
marked, for a glomerulus actually at rest has no space between the glomerulus
and the capsule wall, whereas in the right kidney of this animal no less than
7 of the 10 capsules measured contained fluid, though but small in amount.
We may conclude, then, that both Bowman’s capsule and the glomerulus
are distensible structures, and, further, that during activity the glomerulus
does not remain in contact with the capsule wall, all of which strongly
opposes the filtration theory of glomerular activity. These two conclusions
are confirmed by every experiment we have performed.
When we turn to the measurements of the tubules the changes are equally
striking. The external diameter of the proximal tubule is usually unaltered,
but, whereas the resting tubule has no lumen, the tubule after action has a
large lumen (43 per cent. of the total diameter). With the distal convoluted
tubule the case is somewhat different. The total diameter is markedly
increased (to 153 per cent.). The lumen of the resting tubule is 34 per
cent., but that of the active tubule 64 per cent. of the total diameter of the
tubule. Also, the lumen of the active tubule is 2°86 times greater than that
of the resting. Apparently, then, the basement membrane of the proximal
convoluted tubule is practically inextensible with the forces at play in this
instance, whereas that of the distal convoluted tubule is extensible. In
both tubules the cells are distinctly flattened against the basement membrane
as a result of activity.
EE, Comparison between a Resting and a Decapsulated Kidney.
The measurements obtained in an experiment of this character (Experi-
ment 11) were as follows :—
598 Prof. Brodie and Mr. Mackenzie. On Changes in the
Expt. 11.—R. kidney, resting. L. kidney, decapsulated and secreting
freely.
Re L.
pH. H.
Glomeruli and capsules— '
Equatorial diameter ............... 100°4 1120
Polar diameter.) 2 -55-5-se ee 73°6 95:2
Space 2. Nee. slecteseeten. eeeseae ee 3°0 14:6
Hence
Mean diameter capsule ............ 87-0 103°6
M # glomerulus ...... 84:0 89-0
Approximate volume capsule...... 82 139
ws “3 glomerulus 74. 88
F: fluid fees 8 a!
Convoluted tubules—
Proximal. External diameter... 46:0 42:0
TAM CN).s..<05 seavetece 1-4 19-4
Distal. External diameter ... 24:0 28:0
men Seaceese vee 108 176
grm
Weight of R. kidney......... 8-4
< IVa cars betey anaes We 10°6
In this experiment the changes are entirely in the same direction as in
the preceding, and the magnitude of the various changes is also approximately
the same. If anything, the free kidney in the preceding experiment showed
rather greater changes in comparison to the resting than did the decapsulated
kidney of this experiment. The difference is, however, accounted for by the
fact that the diuresis in Experiment 10 was greater than in Experiment 11.
The increase in volume of the capsule is to 170 per cent., of the glomerulus
to 119 per cent. One notable difference is that in this experiment the
external diameter of the proximal convoluted tubule was less after diuresis
than when at rest.
III. Comparison of a Free Kidney with a Free Decapsulated Kidney.
Expt. 35.—R. kidney free. L. kidney free and decapsulated.
1a L.
B. p.
Glomeruli and capsules—
Equatorial diameter ............... 133°2 142-4
Polardiameter™ 2220 eee. 2S 100°8 1120
SIIRKOS: Bose aaances Goawlele Neee Seto eas 15°2 20°6
Glomerult and Tubules of the Kidney accompanying Activity. 599
Hence
Mean diameter capsule ............ 118-0 1271
i pp glomerulus ...... 100°8 106°5
Approximate volume capsule...... . 203 257
55 » glomerulus 128 151
g 73 uglinid’ see: 17 106
Convoluted tubules—
Proximal. External diameter ... 448 48-0
iG ite) cere eas 13°0 19°8
Distal. External diameter ... 33:2 392
ven ies. cere 24:8 29-2
grm.
Weight of R. kidney......... 20°6
ao Dc kidney .2.:::. 19°1
The two kidneys show the general changes of a diuresis in a well-marked
manner. The experiment further shows that the effect of decapsulation is
to cause a relatively greater expansion of both capsule and glomerulus. Also,
the capsule is not so well emptied as in the normally active kidney. The
difference in the dilatation of the convoluted tubules is again in favour of
the decapsulated kidney. This is particularly seen with regard to the lumen
of the proximal convoluted tubule. Whereas the ratio of the external
diameter of the first convoluted tubule of the decapsulated kidney to that
of the free kidney is 1 to 1-07, the ratio of the lumina is 1 to 1°53.
Hence we may conclude that decapsulation results in an increased
distension of all the cortical parts of the kidney tubule when it is thrown
into activity.
In the next group of experiments one of the kidneys was obstructed. The
group comprises three comparisons.
IV. Comparison of a Resting Kidney with an Obstructed Kidney.
Expt. 12.—R. kidney resting. L. kidney obstructed.
R. L
ro H
Glomeruli and capsules—
Equatorial diameter ............... 98-4 130-4
Polar diameter? 25.0.0 ARs 76:0 111-2
PACE: vis sa este ds dete cnsssceracibds 1:2 248
600 Prof. Brodie and Mr. Mackenzie. On Changes in the
Hence
Mean diameter capsule ............ BT 120°8
Hi 3 glomerulus ...... 86:0 96:0
Approximate volume capsule...... 83 220
ye 5 glomerulus 80 etal
a 5. Ai UMA sete. 3 109
Convoluted tubules—
Proximal. External diameter... 44:0 42°8
Buen. es sacsoeeccionae 0-0 19-4
Distal, External diameter ... 25:4 31°8
EUDOSTYS) Meas arn ee se 11:0 21°8
‘ grm
Weight of R. kidney ......... fart
2 Lkidney: ..2..2-2: 109
The general changes are in the same direction as before. Perhaps the
most marked difference between this and the previous kidneys examined is
the large volume of fluid contained within the capsule, and the relatively
small size of the glomerulus. Again, we note that there is no change in the
external diameter of the proximal convoluted tubule, whereas the distal is
extended to 125 per cent. of its resting diameter. As illustrated by the
lumina, a very considerable volume of urine is collected within the tubules,
particularly in the distal tubule.
V. Comparison of a Free Kidney with an Obstructed Kidney.
Expt. 6.—L. kidney free. R. kidney obstructed.
R.
H. BL
Glomeruli and capsules—
Equatorial diameter ............... 99-6 110°8
Polar diameter c..2cs ene aas 17-2 100-0
NPACE + Sanat emacs Meweetenien ee seen: 6:2 16:0
Hence
Mean diameter capsule ............ 88°4 105-4
- i. glomerulus ...... 82:2 89-4
Approximate volume capsule...... 86 146
5 ee glomerulus 69 89
S 4 UAT ee eee 17; 57
Glomeruli and Tubules of the Kidney accompanying Activity. 601
Convoluted tubules—
Proximal. External diameter ... 39:2 39°4
Tiimenk ey eee 6°6 14:0
Distal. External diameter... 22°8 28:2
Mo mmir ema nseee eee <) 12°6 18°4
grm
Weight of L. kidney ......... NSD
im Rekidney te. 7s.8 155
This experiment shows quite clearly the great effect of obstruction upon
the distension of the capsule and accumulation of fluid within the capsule.
Obstruction also causes a distinct further dilatation of the distal convoluted
tubule, and an increase in the lumina of both parts of the tubule.
VI. Comparison of a Free Decapsulated Kidney with an Obstructed Kidney.
Expt. 7.—R. kidney decapsulated. LL. kidney obstructed.
18, L.
p. B.
Glomeruli and capsules—
Equatorial diameter ............... 121:2 130-4
Rolarsdiameterig sash. -ccacho.2+02e0 102°0 Haley
SJ ORGS) aaydckoun. Son Ne neA ee Ha ene ene ane 9°8 20:0
Hence
Mean diameter capsule ............ 111°6 120°8
ha ;. glomerulus ...... 101°8 100°8
Approximate volume capsule ... 174 220
: é. glomerulus 132 128
® ms MMI! Seonoue 42 92
Convoluted tubules—
Proximal. External diameter ... 42:0 41-4
Wunments, sees aero 15°4 17-6
Distal. External diameter ... 28°6 31:0
uma en tee aceeee ns 20°4 ile
grm.
Weight of R. kidney......... 10°7
‘ oiikacmergre eeere 11:0
The results of the measurements in this experiment show that obstruction
of the ureter results in an increased expansion of the capsule of the
obstructed, as compared to that of the free active kidney; this is entirely
due to a greater accumulation of fluid within it. The convoluted tubules
602 Prof, Brodie and Mr. Mackenzie. On Changes mm the
show corresponding differences. The effect as before is mainly felt in the
distal tubule, which shows a somewhat greater expansion. The lumina in
the proximal tubules are greater in the obstructed kidney than in the free
kidney. In this experiment the blood-pressure was rather low, but the
diuresis good.
In all these obstructed kidneys the effect upon the medulla is very marked.
Not only is the pelvis of the kidney greatly distended, but the pyramid is
driven back towards the cortex, and appears very much shrunken. We have
often seen it so contracted as to appear only about a quarter or less of its
normal size. In the sections the collecting tubules are flattened and empty,
the loops of Henle, however, contain fluid, and often appear to be about the
same size as in the normal active kidney. The appearance of the pyramids
is so characteristic that one can at once decide whether or no the ureter of
that kidney had been obstructed in the experiment.
The last group of experiments comprises a comparison of various kidneys
with a kidney which was both obstructed and decapsulated.
VII. Comparison of a Resting Kidney with a Decapsulated and Obstructed
Kidney.
Expt. 13.—R. kidney resting. lL. kidney decapsulated and obstructed.
R. L
B. B
Glomeruli and capsules—
Equatorial diameter ............... 110-4 128-0
Polar, diameter j-ee--4-seepasto-teeceel ase 110°8
DPACO™ «cence nae eet cesarean o 21-2
Hence
Mean diameter capsule ............ 95:0 119-4
eB ‘ glomerulus......... 91°6 ~ 98:2
Approximate volume capsule...... 107 213
a 2 glomerulus 96 118
z a iltdHel seasconos itil 95
Convoluted tubules—
Proximal. External diameter ... 460 49°6
feumiemtieizee ec sseece re 0:0 26-4
Distal. External diameter ... 218 34:4
Lumenicsrse bese. , decapsulated and obstructed......... 2°95 1°85 13 34
In Tables III and IV we give similar figures for the convoluted tubules.
Table III.
Proximal. Distal.
External External
diameter. SEE, diameter. Thame:
|
MRGSOIN DMs. ecuiecsensnetacsese senses sacae 44 °4 0°4 23 °4 9°9
UACUIVEFETCO Hest se ele ete aae ease et aias 45-0 14°8 33 °0 22 °6
Pr CECADStUl Abed ghcncussss-hcaaecna: 46 °0 17°3 33 °2 BP) ce)
By eS ODSULUCLE OMe raact nee nanicasansic: 42 °0 19:0 29-3 20°3
» decapsulated and obstructed... 47-3 22°6 35 °3 25 *4
Table [V.—Ratios.
| Proximal. Distal.
|
| External External
| diameter. Lumen. diameter. Lumen.
MROSUINIG ME Mun enatan ne ersa sa detnduect antes 1°00 1:00 1°00 1:00
ACHIV OUP OCE ere enter nek ass Oseoei ees OL 37 -00 1°41 2°28
PE CE CHDSUL abe lime nema setinieia: scr 1-04 43 °25 1 42 2°24
Ais MODALLUCKEC tee nea vs oceneaewectnee 0°95 47 50 1°25 2°05
» decapsulated and obstructed 1:07 56 50 1°51 2°57
608 On Changes in the Glomeruli and Tubules of the Kidney.
These two tables bring out the following points :—
(1) The external diameter of the proximal convoluted tubule does not
change on activity ;
(2) A large lumen is developed in this tubule during diuresis. It varies
with the degree of diuresis, and is markedly increased by obstruction of the
ureter. Taking the average of all our observations it amounts to nearly 40
per cent. of the total diameter of the tubule;
(3) The distal convoluted tubule is expanded considerably ae 140
to 150 per cent. of its mean at rest); and
(4) The lumen, of considerable size (42°3 per cent. of the total diameter)
even in a resting kidney, is more than doubled, and becomes 69:2 per cent.
of the total diameter.
We may conclude, then, that the first convoluted tubule, ze. that portion
which is subjected to the highest internal pressure, is relatively inextensible
transversely. The second convoluted tubule, on the other hand, is trans-
versely extensible. From a further examination of our sections, we judge
that the proximal convoluted tubules do indicate an extension in the longitu-
dinal direction, but our present methods do not allow us to state this
decisively.* All the results indicate that an internal pressure has existed
during diuresis.
Conclusions.
Measurements of the diameters of the various portions of the renal tubule
in the cat, when at rest and after diuresis under various conditions, show
that Bowman’s capsule, the glomerulus, and the second convoluted tubule are
extensible structures, and are expanded during diuresis. The glomerulus
leaves the capsule wall, a considerable accumulation of secretion being found
between them. The lumina of all parts of the tubule become greatly enlarged.
All the appearances found are explained as resulting from the action of a
high pressure in the fluid secreted by the glomerular epithelium, and are all
in accordance with the propulsor theory of the action of the glomerulus.
* If we may make the assumption that the volume of the cells cf the convoluted
tubule does not alter during diuresis, then the magnitude of the surface areas of the
cells in a transverse section of the tubule gives us an indication of any change in length.
If, for this purpose, we examine the results of Experiments 10, 11, 12, and 13, where we
have direct comparisons of active with resting kidneys, we find that in all instances the
proximal convoluted tubules are markedly stretched longitudinally. In Experiments 10
and 13 there is considerable shortening of the distal convoluted tubules, and in
Experiments 11 and 12 slight shortening. In Experiments 10 and 13 the blood-pressure
was high and the diuresis good. In Experiments 11 and 12 the blood-pressure was lower
and the diuresis only moderate. Hence it would appear that, with a high internal
pressure, this portion of the tubule is shortened, z.¢. tends towards the spherical shape.
Brodie
and Mackenze.
Roy. Soc. Proc., B, vol. 87, Plate
27.
Influence of Carbon Dioxide in Maturation, etc., of Seeds. 609
DESCRIPTION OF PLATE.
Fig. 1.—Microphotograph of Cortex of Dog’s Kidney at Rest. 500.
Fig. 2.—Microphotograph of Cortex of Opposite Kidney after Activity. 500.
Fig. 3.—Cat’s Kidney. Drawing of glomerulus and tubules after activity, showing
dilatation of neck of tubule. x 500.
The Controlling Influence of Carbon Dioxide in the Maturation,
Dormancy and Germination of Seeds.—Part II.
By Franxkuin Kipp, B.A., Fellow of St. John’s College, Cambridge.
(Communicated by Dr. F. F. Blackman, F.R.S. Received March 25,—
Read May 14, 1914.)
CONTENTS.
PAGE
SHINING LYO MB rata lenient cite te ate(ie ae sales nc sZalins sis ss cinvin sninie sa ciceeiscletecletocenctntnaaieee natlimciemcees 609
Section I.—Relation to Temperature of the Inhibitory Effect of Carbon Dioxide
Onn (CSRUNTTE NAO” qoaaesaaboscconere pe oReauac cor aduenoucaccenacoobeDanacrabasoa 610
5 II.—Relation to Oxygen Pressure of the Inhibitory Effect of Carbon
DroxideqonkGeuminat On seanscntcecea-cessdsscerssemec ces smemeteeccecasecseece 612
3 III.—Carbon Dioxide asa Factor in the Dormancy of the Maturing Seed
OnkGH ede lamitrsenuseeceitasas sake sadovcansbuseesotis veanenuec gress eoanemsomeneeres 614
(a) Arrested Development of Maturing Seeds not due to Lack
of Moisture. Retarding Influence of the Testa............ 614
(6) Direct Estimations of Carbon Dioxide Content of Maturing
caval, (Grenreaduh ne niaVe ISIEYEG ISlapanguassendosdoobaccoddeasaecoconcsacbonoGaD 616
‘3 ITV.—Contrast of Depressant Action of High Partial Pressures of Carbon
Dioxide with Stimulating Effect of Low Partial Pressures.
Carbon Dioxide considered as a Narcotic Agent ...........0.cese000+ 618
es V.—Influence of Carbon Dioxide in enforcing Dormancy in certain
Seeds which do not naturally have along Dormant Phase. Seeds
OLPENCVECMBRASIUCENSUS) seaah aacevettoe atte cetese sede dee eter acacia nee 620
59 V1.—Biological Importance of Dormancy in Moist Seeds..................... 622
» WII.—Summary and Conclusions ............... a eefeleiisele stcenlerineci ae scenes 623
Introduction.
In the first part of this paper the influence of carbon dioxide in
inhibiting the germination of moist seeds was described. The results
obtained are summarised on pp. 623-625 of this paper.
In the present paper the relation of this inhibitory effect of carbon
dioxide to temperature and oxygen supply is first to be examined, and
then will be studied further narcotic or inhibitory effects of CO». as
610 Mr. F. Kidd. The Controlling Influence of
exemplified in the natural inhibition of maturing seeds in the ovary, and
the artificial prolongation of the dormant life of seeds which cannot survive
naturally unless germination occurs soon after ripening.
Section I1—The Relation to Temperature of the Inhibitory Effect of Carbon
Dioxide on Germination.
A large number of experiments were conducted to determine this rela-
tion. Brassica alba seeds were used. The result would appear to establish
the conclusion that at low temperatures inhibition is caused by very small
pressures of COs, while conversely at high temperatures high pressures of
CO, are necessary to maintain continued dormancy. It would seem
probable that this relation to temperature is significant in natural seasonal
conditions. The technique in these experiments was the same as before
described, the details of each experiment in full are unnecessary and a
summary of the results obtained is given. The usual retardation effects
were observed throughout, but the numbers in the table indicate only the
final total germination out of 20 seeds.
Table I—Total Number of Germinations with 20 Brassica alba Seeds in
various Percentages of CO: in Air at different Temperatures. Compiled
from 43 experiments.
|
Percentage of COs ...seeseecseseeee: o|2|4 1] 6] 9 |12| 15 | 18] 24/ 80 | 36} 42
PO. is| 2| 0
7° 0) PS | Ep oe
10° Te ee ees fie | O
17° 20 | 20 | 20| 20/18 |12| 3] 2| o
20° 20 | 20 | 20 | 20| 20 | 20|—|17| 4] 3| 2] o
25/ 20 | 20 | 20 | 20 | 20 | 20| 20] 20| 19| 19] 14] 7
The temperatures 10° C., 20° C., and 25° C. were maintained accurately within a variation of
05°C. The temperature of 3° C. obtained with melting ice varied to the extent of 1°C. The
other two, 7° and 17° C., are averages of outdoor and indoor winter temperatures. The experi-
ments were continued till no more seeds germinated.
It is necessary to consider the possibility of these results being due not
to a decrease with rising temperature in the effectiveness of pressures of
CO: in causing inhibition, but to an increase of oxygen stimulus caused
by an increased permeability of the testa under the action of higher
temperatures.
The following series of experiments were therefore conducted with
Brassica alba seeds from which the testas had been carefully removed.
In these experiments with bare embryos, it was difficult in the early
Carbon Dioxide in Maturation, etc., of Seeds. 611
stages to tell by eye whether germination had begun or not. The bare
embryos in the first stages of germination could not be differentiated, but
had to be described as either all germinating or all not germinating. In
order to bring the results given in the following Table more into relation
with those formerly obtained, small figures have been inserted to express
the relative condition of growth at the end of the experiment, and the
delay in germination as compared with the controls.
Table II.—Total Number of Germinations with 20 Bare Embryos of Brassica
alba Seeds in various Percentages of COs at different Temperatures.
Compiled from 14 experiments.
Control |
Percentages of CO,......... 0 5 10 20 30 40 50
5° C. 20 16 8
16 20 2015 20,5 20, 20,
20 20 2015 2016 2015 20s 20,
Ten per cent. of oxygen was present in each case. The temperatures 16°C. and 20° C. were
maintained accurately within a variation of 0°5° C. In the case of the experiments at 5° C.
the temperature was less accurately controlled, being obtained by melting ice.
It will be seen from the foregoing Tables that a rise of temperature of
10° C. necessitates roughly the presence of three times as high a partial
pressure of CO: to cause inhibition. Thus in Table I at 10° C. no
germinations occurred with CO, pressures above 12 per cent., while at
20° C. germinations occur up to 36 per cent. Similarly at 7° C. no
germinations occur above 6 per cent., while at 17° C. germination pro-
ceeds with pressures up to 18 per cent. It must be remembered that the
actual partial pressures of CO» in the tissues of the embryos is probably
higher, especially where the testa remains intact, than the values
expressed in the tables for the partial pressures of CO: in the atmospheres
used.
The result of this series of experiments, both with whole seeds and with
bare embryos, thus clearly indicates that a rise in temperature necessitates
an increase in the amount of CO2 necessary to produce inhibition in the seeds
of Brassica alba. Conversely, a fall in temperature reduces the necessary
amount of CO. to cause inhibition.
‘This relation of carbon dioxide inhibition to temperature may be
emphasised. In the case of drugs acting chemically on the protoplasm
the expectation is that their action will be more effective at high than
at low temperatures. Here with carbon dioxide the reverse result has been
612 Mr. F. Kidd. The Controlling Influence of
obtained. The fact must be borne in mind that we are dealing in this case
with a gaseous agent more soluble at low than at high temperatures. This
implies that to maintain in solution in the tissues the same concentration of
carbon dioxide at a low temperature as at a high temperature necessitates a
greater partial pressure of CO, in the atmosphere at the low temperature.
Further work upon this relation of carbon dioxide inhibition in seeds to
temperature is needed.*
Section Il.— Relation to Oxygen Pressure of the Inhibitory Effect of Carbon
Dioxide on Germination.
The presence of the testa between the embryo and its gaseous environment,
as a membrane, only permeable with some difficulty, will, as has been pointed
out, cause (1) a reduction in the amount of oxygen reaching the embryo,
and (2) a relative rise in the actual CO. pressure in the embryo tissues.
It has been shown by the removal of the testa that temperature has,
nevertheless, a direct effect in determining the inhibitory value of a given
pressure of COv.
The following experiments were made to determine whether a varying
oxygen supply might not also influence the inhibitory action of carbon
dioxide. A large number of experiments were conducted at the same
temperature, but with varying pressures of oxygen and carbon dioxide in the
atmospheres used.
The testas were not removed in these cases. With a given pressure of
COs, the temperature being fixed throughout, no variation in permeability in
the testa was looked for. It is possible that an increased oxygen supply may -
cause a corresponding increase in the actual CO: pressure in the embryo
tissues. The results show, however, that for the main purpose of the
experiments this possibility may be neglected, as it clearly appears that an
increase of oxygen supply decreases the inhibitory value of any given
pressure of COs, while correspondingly a decrease in oxygen supply intensifies
it, so that with small amounts of oxygen very low percentages of CO2 will
induce complete inhibition.
* In a critica! consideration of the actual pressures of CO,in the embryo tissues at any
temperature we should-have to take into account not only the external partial pressure
of CO,, but also the rate of CO, production in the tissues and the rate of the escape of
this CO, from the tissues by diffusion. Roughly, in relation to different temperatures,
these two processes tend to cancel one another, and their combined effect to give the
same value at all temperatures. In the above experiments no account has, therefore, been
taken of any change with temperature of the rate of CO, production in the tissues or of
the rate of diffusion from the tissues.
Carbon Dioxide in Maturation, etc., of Seeds. 613
Table I1I].—The Effect of Decreased Partial Pressures of Oxygen on Carbon
Dioxide Inhibition in Brassica alba Seeds. Small amounts of COs are
sufficient to cause inhibition if little oxygen is present.
peer Germinations out of
Atmosphere of Sarees dioxide, and SOS eaiS ee Rilo eatiReial Bleseseronet
DINDOT NEOs. remainder which
finally germinated
pee) fel) Garbon' dioxide! | and |) gra’ | “en | sth | Om temoval'tojair.
ygen p Bes. percentages. day. | day. | day. | day.
per cent. per cent. |
21 (air) 0 (air) 18 20 20 | 20
8 0 13 18 18 20
8 1 13 19 19 20
8 3 15 15 18 20
8 6 — 12 13 16 All
4, 0 6 18 18 19 All
4 1 — il 1 3 All
4 6 — — — 9 All
Average temperature, 14° C.
It will be noticed from the preceding Table that the effect of decreasing
the oxygen supply is to intensify the inhibitory action of carbon dioxide.
Thus, with a decrease of oxygen to 8 per cent., the inhibitory effect produced
by 6 per cent. of carbon dioxide is very marked. With a decrease of oxygen
in the atmosphere to 4 per cent., complete inhibition is produced by 6 per
cent. of carbon dioxide.
Here again it must be remarked that this relation may very likely be
significant in many cases of delayed germination under the influence of CO:
in natural conditions.
Table IV.—Effect of various Partial Pressures of Oxygen on the COs:
Inhibition of Germination in Brassica alba Seeds. Total number of
germinations obtained out of 20 seeds.
Percentages of CO; ......... 0) © 12 15 18 21 24 27 30
Oxygen 5 per cent. ......... | @ 4, 0) 0) 0 0 0) 0
eo) FON eckha tis 20 18 7 10 3 il 0) 0 (0)
oy ls O) Fea eeEee 20 20 20 15 10 2 0 (0) (a)
PORES, Alerey, LACE): BA) l> PA0y |e C4O> pen) | edt} I 3 0 0
» 380 Sk Hilttccch ates. 20 20 20 20 _ 11 5 2 1
|
Average temperature 16°7°C.; extremes 13-18°C. The atmospheric residuum is N, in these
experiments.
614 Mr. F. Kidd. The Controlling Influence of
It will be seen that the amount of oxygen present has a definite effect
upon inhibition by COz. Where there is only a pressure of 5 per cent.
oxygen, complete inhibition is obtained by 15 per cent. COs, but, with
30 per cent. oxygen present, as much as 30 per cent. CO is scarcely
sufficient at the temperature used to cause inhibition.
The result of these experiments, therefore, indicates that a rise in the
partial pressure of oxygen within the limits experimented on necessitates an
increase in the amount of CO2 necessary to produce inhibition in the seeds of
Brassica alba. Conversely, a fall in the partial pressure of oxygen reduces
the necessary amount of CO: to cause inhibition.
Section Il1I—Carbon Dioxide as a Factor in the Dormancy of the Maturing
Seed on the Plant.
(a) Arrested Development of Maturing Seeds not due to Lack of Moisture—
Retarding Influence of the Testa——The maturation of the seed in normal
conditions has certain features upon which it is desirable to dwell briefly.
The growth of the embryo proceeds continuously after fertilisation. In
some cases it quickly reaches an advanced stage, and the radicle, plumule,
and cotyledons may be formed very early. This growth, moreover, appears
to resemble in some respects the growth which takes place subsequently,
after germination, but in others it has the appearance of partial inhibition,
the radicle apparently being not free to sprout as in germination. This
appearance of inhibition increases in the cases of most seeds, until at the
stage of complete maturation growth is apparently arrested or suspended.
That there is some restraining cause tending to prevent growth present in
the seed during the series of changes which is producing maturation may be
proved, as in the experiments following, by the fact that the embryo, often at
a comparatively early stage, though the seed be far from ripe, can be caused
to sprout if removed to air.
The following experiments were conducted in order to show that neither
lack of water nor any physiological insufficiency in the embryo can be
considered as the cause preventing the still maturing embryos of beans and
peas from sprouting, and so becoming cases of viviparity :—
(1) Two lots, 10 peas and 10 beans, were taken from pods which were still
perfectly green and hardly yet fully swelled. These two lots were set to
germinate at 20° C. on damp sand, with the result that all the seeds
germinated perfectly. From these experiments it is clear that, in the case
of the bean (Vicia faba) and of the pea (Pisum sativum), for some con-
siderable period before the natural drying process commences, and while the
growth of the pods is continuing, the seeds, if removed and placed in
Carbon Dioxide in Maturation, etc., of Seeds. 615
germinating conditions, are capable of immediate germination. In this and
similar experiments it was noticeable, especially in the case of peas, that
removal of the testa greatly increases the rate of this germination. The
following experiment was typical :—
Table V.—Increased Rate of Germination in Maturing Seeds of Peas
when Testa is removed.
| Germinations.
Description of seed. Remarks.
| 3rd day. | 8th day.
I |
Peas fresh from the pod (10 with 0 4, The testas of the six not growing
testa) were removed on the 8th day. |
All these six then sprouted
within two days. |
Peas fresh from the pod (10 without 3 8 }
testa) | |
(2) Further experiments to test the power of the embryo of the ripening
bean and pea, before drying has commenced, to grow without the addition of
moisture, were necessary. To this end 10 bean embryos taken from seed in
immature condition were placed in glass tubes closed at both ends with
bored rubber corks. They were placed at such distances as to avoid
contact with each other.
In six days the radicles of all had sprouted; similar results were obtained
with embryos taken from immature pea seeds.
These experiments were repeated another year with confirmatory results.
The bare embryos germinate readily in the above conditions. In parallel
experiments made with whole immature seeds, the presence of the testa still
intact was found to retard sprouting constantly. This retarding effect of the
testa was more marked in these cases where no water was added to the green
seeds from the pods than in the experiments above, in which such seeds were
germinated in the ordinary way on damp sand. In connection with this
action of the testa it is of great interest to find that Guppy, in a recent
book containing the results of a wide series of studies upon seeds, remarks
that “it is noteworthy that the viviparous habit is associated with the
absence of seed coats.”
(3) Experiments with germinating beans after complete air-drying in the
laboratory showed that, at the moment of sprouting, these seeds might
actually contain less water than they did when originally removed from the
pod. These experiments were conducted both with whole seeds and with
\
616 Mr. F. Kidd. The Controlling Influence of
the embryo alone. The following are representative examples taken from a
series of experiments :—
Table VI.—Showing that Beans germinated after Complete Air-drying may
actually contain at the moment of sprouting less water than they did
when originally removed from the fresh green pod in the last stages of
maturation before drying on the plant had commenced.
Weight of the same 10 beans at
the moment of sprouting during
germination on damp sand after
complete air-drying.
Original weight of 10 beans when
| removed from fresh pods before
natural drying had commenced.
grm. grm.
Whole seeds ......... 27 °7 22 °6
rare OR: Sh Ae 24-1 21°6 |
| Embryos alone ...... 22-0 20i1 !
|
From these experiments it would appear, therefore, that neither lack of
water nor any physiological insufficiency in the embryo can be regarded
as the factor limiting germination in the maturing seeds of peas and beans.
Finally, the action of the testa as a retarding influence on germination has
to be noted. In addition to experiments already given with seed still
immature, the following experiments were made with dried seeds :—
Table VII.—Retarding Influence of the Testa in Germination of Dried Seeds.
Water uptake Germinations.
after 24 hours
Description of seeds. in percentage of |
original Ist | 2nd | 3rd | 4th | 5th | 9th
dry weight. day. | day. | day. | day. | day. | day.
|
Peas 8 days dried in air after removal |
from pod, temp. 18-14° C.—
LO with testas .7..-...0.-..s0.-sreees 139 0 0) 1 5
10 without testas .................:-+-| 112 | O 0 5 i
| Dry beans—
LVO\with) testasweesc.cds--het-o-- sass 116 0 0 0 4 9
10 without testas..................-.. 119 (@) 1 3 8 10
Dry beans—
1O-with testas ../.....cc0.s020s5e-:es 100 0 (0) 1 3 / — 8
10 without testas..................... 116 O10 | Oo — | 10
In the above Table the retarding influence of the testa in the germination ‘
of seeds after drying is well marked.
(b) Direct Estimation of the COQ Content of Maturing and Germinating
Seeds.—An enquiry is strongly suggested as to how far the non-germination
Carbon Dioxide in Maturation, etc., of Seeds. 617
of the maturing seed, while still upon the parent plant, may be due directly
to CO: inhibition or narcosis. In order to obtain evidence here an endeavour
was made to ascertain the actual CO2 content of ripening seeds. The method
adopted was suggested by Dr. F. F. Blackman, for whose advice and direction
during these researches I am deeply indebted. The technique of this method
for determining the amount of CO2 present in the tissues of seeds was as
follows :—
Two lots of material of equal weight were taken in each experiment.
One lot was crushed to thin paste in a mortar and left exposed to the air for
40 to 80 minutes. It seemed from experiments that this time was sufficient
to allow the escape of the CO: present in the tissue mash. A known
quantity of baryta was then added and a titration made with HCl. The
second parallel lot was crushed immediately under an equal quantity of
baryta and a similar titration made.
The difference between these two readings invariably showed that more
baryta had been neutralised where the tissues had been crushed immediately
in contact with it than where the tissue was first exposed for some time to
air after crushing to a mash. These differences were taken as roughly
expressing the relative CO, contents of the tissues used in these experi-
ments.
The results obtained in a series of experiments made by this method to
ascertain the CO, content of maturing peas and beans from fresh green pods
and of the same seed during its drying in laboratory air, are given in the
following table :—
Table VIII.—The CO, Content of Maturing Peas (Pisum sativum) and Beans
(Vicia faba) when removed fresh from the Green Pod and during the
first few days of drying.
Cubic centi-
HO per, |. eee ek |) ato CO,/H_O in
| | Grammes of
See an LE 100 grm. {0s isi f tissues of seed.
of seed. | pee
| seed.
(ees fresh fromithe pod... /i.-..2-..0d2/--.se22 50 54 108/100
Peas after 4 days in laboratory air ............ 22 145 660/100
| Beans fresh from pod ............-.--.2.-+-000+ 60 51 85/100
| Beans after 1 day’s drying in laboratory air 56 | 46 82/100
Beans after 4 days’ drying in laboratory air | 51 41 80/100
In comparison with the above results the following Table gives those
obtained in a second series of experiments made to determine the CO,
618
Mr. F. Kidd. The Controlling Influence of
content of similar seed during ordinary germination on damp sand after
complete air drying in the laboratory :—
Table IX.—The CO: Content of Beans (Vicia faba) and Peas (Pisum
sativum) while germinating.
| |
Grammes of ule oat
ee H,0 per Ratio CO,/H,O in
Description of seed. 100 erm. of aes per ; (usudeieh acadl Growth.
seed Sree
: seed.
Peas after 18 hrs. germinating | 67 64 96/100 None
55 3 20. 5s 67 41 61/100 None
“ » 39 3 70 43 62/100 Sprouting
3 »» 64 3 70 39 55/100 3
* 5 BH 65 16 24/100 :
Beans after 24 hrs. germinating 58 20 34 °5/100 5
Beans after 7 days in germina-
ting conditions .................. = = 41/100 ~
Beans after 5 days withouttestas
in germinating conditions ... = | = 16 °5/100 3
|
|
The experiments lead to the conclusion that in the maturing seed, in the
case of beans and peas, the CO: content of the tissues is higher than that under
which actually germination takes place. In short, so far as these experi-
ments have gone, it would seem that where the CO: content of the tissues is
above a certain point germination does not occur and that the Coe content
must fall below this point before germination takes place.
Section 1V.—Contrast of Depressant Action of High Partial Pressures of
Carbon Dioxide with Stimulatory Effect of Low Partial Pressures.
Carbon Dioxide considered as a Narcotic Agent.
From the experiments already described it is definitely shown that the
phenomenon of non-germination induced in the seed by CO: is one of
temporary inhibition resulting in a condition strikingly similar to that of
narcosis. The interesting question therefore presents itself as to how far
this depressant action of carbon dioxide can be regarded as true narcosis.
Looking back, in the first place, through the history of previous work, it
has to be noticed that the following results have been recorded as to the
effect of carbon dioxide on the growth activity of plants :—
De Saussure (3) in 1804 found that an atmosphere containing 8 per cent.
COs restrained the growth of peas. Montemartini(4) found that over 7 per
cent. CO2 depressed the growth activity in the roots of peas. Chapin(5) in
1902 confirmed this. Bohm(6), Dr. Drabble(7), Prof. Farmer(1), and
Carbon Dioxide in Maturation, etc., of Seeds. 619
Brown and Escombe (2) have also conducted experiments tending to show
the restraining effect of carbon dioxide on growth. Dr. Drabble and
Miss Lake in 1905 demonstrated the stimulation effect of small partial
pressures of CO2, observing that the growth in the length of pea roots
was more rapid in 4 per cent. CQ, than in air and than in percentages
greater than 7 per cent. of CO».
Here it will be observed that there are two classes of effects recorded; an
effect of retardation by higher percentages, and an effect of stimulation by
lower percentages of COz. The stimulatory effect of small doses is a general
property of narcotic agents. A further series of experiments was therefore
arranged to test the effect of CO. in various proportions below the inhibitory
percentage on the germination and growth of Brassica alba and Hordeum
vulgare. The results* obtained with Brassica alba are shown in the following
Table.
Hordeum vulgare gave similar results.
Table X.—Results obtained in Growth of 10 White Mustard (Brassica alba)
Seeds under increased Partial Pressures of COs, showing the Stimulatory
Effects of Low Percentages, rising to a Maximum and then declining
towards Inhibition.
| | |
| Increase in Average length
| Percentage of CO, weight expressed of growth at
in the atmosphere in | in percentages termination of
each case. of original experiment
weight of seed. | in centimetres. |
|
0 233 3°8
2 16-0 4°0
3 34‘0 4-4
4 23-0 4°3
| 5 9-0 3°5
0 8-0 2-0
(25 per cent. CO, gives
complete inhibition)
This experiment was conducted in a dark room, Average temperature, 16°5°C.
In the foregoing Table it will be observed that the first effect of carbon
dioxide is one of stimulation in low percentages. This increases to a
* The rate of germination was not increased by the low percentages of CO, in this
experiment, but as has been shown in the case of beans and peas in Table IX, the actual
CO, content of the seeds is high and falls from an initially inhibitory value as germination
proceeds. We should not expect, therefore, small doses of CO, in the atmosphere to have
a marked stimulatory effect, if any, upon the rate of germination, though their effect
upon growth after the escape of the initial high partial pressures of CO, in the seeds is
clear.
VOL. LXXXVII.—B. 3A
620 Mr. F. Kidd. The Controlling Influence of
maximum which, at the temperature used, is obtained at about 3 per cent. or
slightly over, and then declines again through a restraining effect to complete,
inhibition. oT
We appear, therefore, to have in view results confirmatory of the
hypothesis that we are here dealing with the effect on. germination and.
growth of a true narcotic agent, and that the results induced by CO2 in the,
resting seed are a phase of narcosis.
Section V.—Influence of CO2 in Enforcing Dormancy in Certain Seeds which’
do not Naturally have a Long Dormant Phase. Seeds of Hevea
brasiliensis. ome,
A considerable amount of work has been done in the past—work which is,
well summarised by Becquerel(8)—upon various effects produced in dry
seeds by sealing them in various gases and vapours, including CO: Becquerel:
discounts the value of part of this work on the ground that it has been
conducted on seeds with impermeable testas, so that the gases used could not
be considered to have reached the plant embryo.
In a number of experiments conducted during this inquiry on seeds with
naturally permeable testas, and on rapidly deteriorating seeds in which the
testas may be assumed to be at least partially permeable, carbon dioxide was
found in nearly all cases to have certain definite effects, such as might have
been expected from the foregoing experiments conducted upon wet seeds in
germinating conditions.
The results of this work, which is still in progress, have not yet been
correlated, but one aspect of them may be referred to here, as bearing directly
upon the central problem discussed in this paper.
One of the most rapidly deteriorating seeds is that of Hevea brasiliensis.
In planting in the tropics it is found that it is always desirable to put the
seed in the ground within a fortnight, and Mr. C. Curtis, late director of the
Botanical Gardens, Penang, from whom the seeds used were obtained, writes
that even in such circumstances 70 per cent. germination is considered good.
This rapid deterioration of the seed has been a difficulty in the recent
extension of rubber plantations, and the question of the best conditions for
preservation in packing and export has been an important one, leading to
practical research. The seeds are at present usually packed in ground
charcoal and ashes. Their size is about that of an average acorn or larger.
They have easily permeable testas and a high water content,and while living
they were found to be respiring very rapidly. They were also found to be:
very intolerant of drying. The seeds in the experiments considered in this’
research were enclosed in hermetically sealed flasks under various conditions,
Carbon Dioxide in Maturation, etc., of Seeds. 621
and it was found as the outcome of a number of experiments that when they
were sealed in the proportion of 40 to 50 seeds to 1200 cc. of air the
following results-were obtained :—(1) A partial pressure of CO2 of 40-45 per
eent. was created in the flasks by the life processes of the seeds, and (2)
there was a marked prolongation in their period of vitality.
In the following. Table the results of two experiments are given. The
imported seeds, when received in this country, were necessarily some weeks
old. The temperature at which germination tests were conducted was 27° C.
in a thermostat :—
Table XI.—Showing prolonged Dormancy of Hevea brasiliensis Seeds
sealed in flasks as described. Flasks opened after 50 days. A test
germination, begun at the time of receipt of the seeds, gave 40 per cent.
germinations.
Analysis of atmosphere on
opening flasks after 50 days. Percentage of
How kept during 50 days. t germinations after
| 60 days.
cO;.
|} | Ne
! }
|
‘ | per cent. | per cent. | per cent. per cent.
Experiment 1—
50 seeds in air in 1200 c.c. sealed _ 45 L3y [Lee 5e 40 (good plants)
flasks |
50 seeds in air in 1200 c.c. open flask SS ol) | = 8
_ 50 seeds in airin commercial packing = — — |. 16
as sent from tropics ; |
Experiment 2— rik |
20 seeds in air sealed in 500 c.c. flask 40 | 4:0 56 40 (good plants)
20 seeds in nitrogensealed ind00c.c.| 41 | 1°0 58 25 is
flask | |
20 seeds in air in 500.¢.c. open flask | — | — i 0
The first of the above experiments took place over the months of November
and December. The flasks were kept in a temperature varying from 10° to
15? C.
The second experiment took place during December and January. The
flasks were kept in the laboratory, the temperature varying from 18° to
13° C. There was considerable internal pressure when the flasks were opened
in both experiments. ;
In a third experiment the period during which the seeds were kept from
date of importation was prolonged to 90 days. The average temperature
was considerably higher, the months over which the experiment extended
being September, October, and November. Im this case 10 per cent.
622 Mr, F. Kidd. The Controlling Influence of
germinations were obtained with seeds sealed in air in the proportion
mentioned against nz with seeds kept in commercial packing, nil with
seeds kept in open air, and nl with seeds sealed in nitrogen.*
In the foregoing experiments it will be observed that large seeds enclosed
in permeable seed coats and sealed with a definite proportion of air in an
impenetrable outer envelope were being dealt with. In these conditions,
where the life processes of the seeds resulted in the creation in the flask
of a partial pressure of CO2 of 40-45 per cent. the vitality of the seeds
was markedly prolonged.
A conclusion which Becquerel reaches, as the result of his researches, is
that in all cases of longevity in dry seeds the testas are exceptionally
strong and impermeable. The problem of the dry seed enclosed in an
impermeable or almost impermeable testa has certain striking affinities—
in that gaseous exchange in either direction is hindered or prevented—to
that of the wet seed, though in apparently good germinating conditions,
which does not germinate. But with the former problem we are not at
present directly concerned in this research.
Section VI.—Biological Importance of Dormancy in Moist Seeds.
The seed is a comparatively late arrival in geological time, and the
perfecting of its function has of necessity been a great point in the struggle
for existence amongst plants. A leading cause in the success of the
Angiosperms, as Prof. Seward has pointed out, has consisted in the
efficiency of the arrangements for nursing the embryo. There can be no
doubt that a ruling factor in this efficiency has been the adjustment of
all the life processes of the moist resting seed to the end of attaining a
fit time for germination. It is suggested by these experiments that the
presence of carbon dioxide in the tissues of the embryo acting as a
restraining and inhibiting agent on the life processes of the seed, and as
a dominant factor in relation to the oxygen stimulus, has been utilised
in attaining this efficiency of the latent seed for which fit conditions of
germination have not yet arrived. The various structures of the testa and
its behaviour under different conditions in regulating the gaseous exchanges
* The favourable results obtained in these experiments in prolonging the vitality of
these rapidly deteriorating seeds were greatly in excess of those which are secured by
present commercial methods of packing for transport and import. In experiments on a
large scale the seeds might be simply sealed (in the proportions of air mentioned) in large
carboys, such as are used for the transport of distilled water, covered with wicker or wire
netting. In case of too high an internal pressure, arising from overfilling with seeds, a
simple form of safety valve might be inserted in the sealing.
Carbon Dioxide in Maturation, etc., of Seeds. 623
appear to be important factors in obtaining the necessary adjustments to
natural conditions.
Emphasis may properly be laid on the fact that it is these adjustments of
the moist seed when in apparently suitable conditions of temperature,
moisture, and oxygen supply, while awaiting the fit time for germination,
and not so much the adjustments of the resting dry seed, that have formed the
central problem of seed life in conditions of nature. The maintenance of
latency when the moist seed is in conditions of medium temperature,
oxygen supply, and moisture, has been the problem of the maturing seed
on the parent plant. It has been the problem of a large proportion of
native seeds which fall upon the ground in summer and autumn, but whose
fit time for germination does not arrive till the following spring. It has,
beyond doubt, been the problem also of many species of plants in the struggle
for existence whose chances therein must have often been increased many-
fold by the capacity of their seeds to lie dormant in the ground for indefinite
periods, ready to resume activity with sporadic germination when suitable
conditions arise such as, for instance, occurred in the case of the Brassica alba
seeds of these experiments when the testas became dry or ruptured.
Section VII.— Summary and Conclusions.
Part I—Experiments were conducted showing that the germination of
seeds is retarded or inhibited by high partial pressures of CO» in the
atmosphere. This retardation and inhibition produced by CO2 was shown
to be unaccompanied by injury. The seeds used in these experiments fall
into two classes. In the first class the seeds germinated at once after
removal from the inhibitory CO: pressures (beans, cabbage, barley, peas,
onions). In the second class the inhibition continued indefinitely after the
removal of the inhibitory COz2 pressures, and is terminated only by complete
drying (and rewetting), or by the removal of the testa. In this class a
lowering of the permeability of the testa to gases under the influence of COs
is indicated, a change which would have two results: (1) a reduction in the
amount of oxygen reaching the embryo ; and (2) a relative rise in the actual
COz pressure in the embryo tissues. The condition of prolonged inhibition
after removal to air produced in Brassica alba is strikingly suggestive of the
condition of seeds often met with in nature, the germination of which is
delayed in spite of suitable conditions of temperature and water. The results
obtained in the laboratory with Brassica alba seeds were reproduced in the
soil in natural conditions by CQ: arising from decaying vegetable matter.
The high CO, content of the soil air in these experiments was found to
624 “Mr. F. Kidd. The Controlling Influence of
continue for a considerable period. Attention was called. to the importance
of these facts in agriculture.
Part II—A long series of experiments was carried out to: detente the
relation of carbon dioxide inhibition in seeds to temperature and to oxygen
supply. Low temperatures and low oxygen supply were both found to
increase the. inhibitory value of given partial pressures of CO», while
inversely the inhibitory value of given carbon dioxide pressures diminishes
with a rise of temperature and with a rise of oxygen pressure. The
probable relation of these facts to the dormancy of the moist seed in natural
conditions was pointed out.
The arrested development of maturing seeds on - the sslahe was shown not
to be due to lack of moisture or to any physiological insufficiency. The seeds
in this stage were shown to contain in their tissues more CO. than seeds
normally germinating contain at the moment of sprouting. The presence of
the testa was shown constantly to retard the germination both in seeds taken
from the parent plant before natural drying and in seeds after complete
drying and storing. Attention was drawn to the correlation found to exist
between the viviparous habit and the absence of seed coats.
Carbon dioxide has been considered as a narcotic agent. Previous work on
the action of CO. upon growth has been quoted. The stimulatory effect of
low partial pressures, rising to a maximum with increasing pressures and then
declining to inhibition with higher pressures of COs, has been demonstrated
by experiments with Brassica alba and Hordeum vulgare germinated in
the dark,
In the case of certain rapidly deteriorating seeds (Hevea brasiliensis) the
carbon dioxide naturally produced by respiration of the seeds in a closed flask
rose to 40 per cent. and the presence of this was found to be accompanied by
a marked prolongation of vitality in the seeds. This prolonged vitality was
far in excess of that reached with the present commercial method of packing
these short-lived seeds for export.
When we correlate the results of these different lines of experiment we
seem to get in various directions evidence of the importance of carbon dioxide
pressure as a controlling influence in the biology of seeds. This influence
may be formulated briefly in the following principles :—
(1) The resting stage of the moist seed is primarily a phase of narcosis
induced by the action of carbon dioxide.
(2) Both the arrested development in the case of the moist maturing seed
on the plant, and the widely occurring phenomenon of delayed germination in
the case of the moist resting seed, which does not germinate although in
apparently suitable conditions of temperature, moisture, and oxygen supply,
Carbon Dioxide in Maturation, etc., of Seeds. 625
are related to an inhibitory partial pressure of carbon dioxide in the tissues of
the embryo.
(3) Germination when it takes place is related to a lowering of the value
of this inhibitory partial pressure of carbon dioxide in the tissues.
(4) The inhibitory value of a given carbon dioxide pressure diminishes with
a rise of temperature.
(5) The inhibitory value of a given carbon dioxide pressure diminishes with
a rise of oxygen pressure.
LITERATURE CITED.
1. Farmer, J. B., and S. E. Chandler, “On the Influence of an Excess of Carbon Dioxide
in the Air on the Form and Internal Structure of Plants,” ‘ Roy. Soc. Proc.,’ vol. 70,
pp. 413-422 (1902).
2. Brown and Escombe, “The Influence of varying Amounts of Carbon Dioxide in the
Air on the Photosynthetic Process of Leaves and on the Mode of Growth of
Plants,” ‘Roy. Soc. Proc.,’ vol. 70, pp. 397-413 (1902).
3. De Saussure, ‘Recherches Chimiques sur la Végétation,’ Paris, 1804.
4, Montemartini, “Sulla Influenza di Atmosfere ricche di Biossido di Carbone sopra lo
Sviluppo e la Struttura delle Foglie,” ‘Atti del Istituto Botanico di Pavia,’ 1892.
5. Chapin, P., “Einfluss der Kohlensaure auf das Wachsthum,” ‘ Flora,’ 1902, p. 348-379.
6. Bohm, Jos., ‘Sitzungsberichte der Wiener Akademie,’ 1873.
7. Drabble, Dr. E., and Miss Lake, “On the Effect of Carbon Dioxide on the Geotropic
Curvature of Roots of Pisum sativum, L.,” ‘Roy. Soc. Proc.,’ B, vol. 76, pp. 351-358
(1905).
8. Becquerel, “ Recherches sur la Vie Latente des Graines,” ‘Ann. Sci. Nat., Bot.,’ ser. 9,
5, pp. 193-307 (1908).
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JOHN LUBBOCK, BARON AVEBURY—1834-1913.
Tue. first Lord Avebury, for many years better known as Sir John Lubbock,
died on May 28 last, in his 80th year. He was the eldest son of the third
Baronet and Harriet, daughter of Captain Hotham, of York. He was
educated at Eton, but left at an early age to join his father in the family
bank. He married firstly Ellen the eldest child of Peter Hordern, and some
years after her death, in 1879, Alice Augusta Laurentia, daughter of the late
General A. A. Lane-Fox Pitt-Rivers, and grand-daughter of the second Baron
Stanley of Alderley. In 1865, he succeeded his father as fourth baronet, five
years later he became Member of Parliament for Maidstone, and held this
seat until 1880, when he was elected representative of the University of
London. This seat he held until 1900, the date when he was removed to
“ another place,” as Baron Avebury.
Lord Avebury took an active but restricted part in politics. His most
prominent efforts were directed to the establishment of Bank Holidays, but
he devoted much time and attention to educational questions and social
reform. Without having had a University training he was yet peculiarly
fitted to be a representative of a University, being a man of wide culture
as well as a very competent man of business. For many years he was
head of the great banking company, Robarts, Lubbock and Co, and by
his tireless activity and ceaseless care for detail, he became a very prominent
man in City circles. This attention to detail and his knowledge of procedure
made him an admirable President ; and, indeed, he seems to have presided
over nearly every scientific society and countless mercantile associations.
At various dates he was President of the British Association (Jubilee Year),
the Entomological Society, the Ethnological Society, the Linnean Society, the
Anthropological Institute, the Ray Society, the Statistical Society, the African
Society, the Society of Antiquaries, and, the Royal Microscopical Society.
He was also the first President of the International Institute of Sociology,
the President of the International Association of Prehistoric Archeology, the
International Association of Zoology, the International Library Association,
the London University Extension Society, and the first President of the
Institute of Bankers, President of the London Chamber of .Commerce, and
of the Central Association of Bankers.
For eight years he was Vice-Chancellor of the University of London, and
he was also Principal of the Working Men’s College. He sat on many a
Royal Commission, and left his mark on those on the Advancement of Science,
on Public Schools, on International Coinage, on Gold and Silver, and on
Education. He was perhaps less happy as President of the Committee
which selected the designs for our present coinage.
il Obituary Notices of Fellows deceased.
At the time of his death, Lord Avebury, although he retained a house in
London, had given up his house in St. James’s Square, and he died at
Kingsgate Castle, Kent. Another of his country residences was High Elms,
Down, and it may have been the association of Darwin and Avebury at
this small Kentish village that first attracted Lord Avebury’s attention to
natural history,
One of his first books, and perhaps one of the most stimulating, was ‘The
Origin of Civilisation and the Primitive Condition of Man,’ now in the sixth
edition, a book which aroused interest and research in the past in many
quarters. It was characteristic of him when he had to select a title for his
peerage to choose that of Avebury, the preservation of whose prehistoric
remains he had taken so large a part in securing. At the time of his last
illness he was engaged in revising and partly rewriting a seventh edition of
his well-known ‘ Prehistoric Times.’
Without being a great researcher, Lord Avebury took a very prominent
part in encouraging the research of others. Of his more scientific works,
perhaps his monograph (published by the Ray Society) ‘On the Collembola
and Thysanura’ has proved most useful; for a long time it was the authori-
tative work on these lowly insects, and still is so, especially with regard
to the Collembola, whose distinction from the Thysanura was first recognised
by the author. But many of his other works passed into numerous
editions: ‘ British Wild Flowers, considered in Relation to Insects, reached
the sale of 11,000 copies ; ‘ Ants, Bees, and Wasps’ passed into the seventeenth
edition ; and his works on ‘ Seedlings’ and on ‘ Buds and Stipules’ contained
much that is valuable and well worthy of record.
He wrote two geological works which are still used with profit by students
of the Universities; one on ‘The Scenery of Switzerland, and the other,
published ten years ago, on ‘The Scenery of England, and several treatises
on more strictly economic lines. His works on Coins and Currency, on
Free Trade, and on Municipal, and on National Trade, occur to one’s
mind. But apart from these more or less technical publications, Lord
Avebury had a genuine “flair” for writing books which the public want.
Both parts of ‘The Pleasures of Life’ sold over 200,000 copies, and
Part I over a quarter of a million, besides being issued in no less than
forty foreign editions. ‘The Use of Life’ and ‘The Beauties of Nature’
were hardly less successful, and everyone will remember his “ Hundred Best
Books.”
As the foregoing will show, Lord Avebury was a man of singularly
diversified activities and extreme width of interest. That he should find
occasion in the middle of a busy business career to do the work he did
is indeed amazing, but he was precise and very business-like, and knew
how to make the most of his time.
He had after his name an alphabet of Honorary Degrees and memberships
of Learned Societies. It need hardly be said that he was covered with
honours too numerous to enumerate. He was Lord Rector of the University
Philip Lutley Sclater. il
of St. Andrews, Trustee of the British Museum, and Foreign Secretary to the
Royal Academy. He served five distinet periods on the Council of the Royal
Society, the last being in the year 1906-7, and was three times Vice-
President. He was Commander of the Legion of Honour, and held the
German “Ordre pour le Mérite.”
As IB Ss
PHILIP LUTLEY SCLATER—1829-1913.
Partie LUTLEY SCLATER was born in November, 1829, at Tangier Park, in
Hampshire, where his father, Mr. William Lutley Sclater, then resided, though
he shortly after moved to Hoddington House, another estate in the same
county, not far from the old home of Gilbert White, where his boyhood was
passed.
In 1842 he went to Winchester College and was elected a scholar of Corpus
Christi College, Oxford, in 1845, but being under age was not called into
residence at the University until the following year. At Oxford he devoted
his studies chiefly to mathematics, but at the same time he occupied much of
his spare time in the pursuit of natural history, his speciality, as in after life,
being ornithology. While at Oxford he was fortunate in becoming acquainted
with H. E. Strickland, and at his house he met John Gould, shortly after the
return of the latter from Australia. It was from them that he received his
first serious instruction in ornithology, and it was during his Oxford days that
he commenced his collection of birds.
In 1849 he took his degree, obtaining a first class in Mathematics and a
pass in Classics, but he remained for two years longer at college before
proceeding to his M.A. degree. During this time he also studied modern
languages and became familiar with French, German, and Italian, spending as
much of his time as he could spare on the Continent. At Paris he made the
acquaintance of Prince Charles Bonaparte, at whose house he was a constant
visitor, and thus he received a further stimulus in his favourite pursuit of
ornithology.
In 1855 Sclater became a Fellow of Corpus Christi College, Oxford, and
was called to the Bar by the Honourable Society of Lincoln’s Inn and went
on the Western Circuit for several years. In 1856 he visited America, in
company with a friend, and attended the American Association for the
Advancement of Science, at Saratoga, after which they proceeded to Niagara
and the Great Lakes, and on foot to the upper waters of the St. Croix River,
thence descending in a birch-bark canoe to the Mississippi. They finally
returned to Philadelphia, where Sclater spent some time studying the fine
collections at the Academy of Natural Sciences and meeting John Cassin,
iv Obituary Notices of Fellows deceased.
Joseph Leidy, John le Conte, and other well- agen naturalists, returning
to England about the end of the year.
He now took up his residence in London, continuing his studies i in natural
history and also practising at the Bar. He was a constant attendant at the
meetings of the Zoological Society of London, of which he had been previously
elected a Fellow, and in 1857 became a Member of the Council. In 1859
Sclater, in company with his friend E. C. Taylor, made an expedition to Tunis,
visiting the breeding places of the vultures, eagles, and other Raptores and
making considerable collections.
About this time Mr. D. W. Mitchell, who had been Secretary to
the Zoological Society, was appointed to superintend the new Jardin
d’ Acclimatation in Paris; thus the post became vacant, and Owen and Yarrell,
influential members of the Council, induced Sclater to apply for it, and at the
Anniversary Meeting in 1859 he was unanimously elected. On his appoint-
ment he found that a considerable re-organisation of the Society’s affairs was
necessary, the ‘ Proceedings’ and ‘Transactions’ were sadly in arrear, and
the gardens themselves were much neglected. He at once set to work to
reform these matters, and as a result the prosperity of the Society vastly
increased. The number of Fellows was augmented from about 1700 in 1859
to above 3000 when he resigned his post in 1902, and, similarly, the income
rose in the same period from £14,000 to £30,000 and both the buildings in the
Gardens and the offices in Hanover Square were replaced by much more
suitable and commodious structures, the library also received great attention
and now became an important feature of the Society. From 1874 to 1876 he
became private secretary to his brother (then the Right Honourable Sclater-
Booth, M.P., and afterwards Lord Basing), when he was President of the
Local Government Board in Mr. Disraeli’s Administration.
The British Ornithologists’ Union was established in 1858 for the study of
general ornithology and Sclater was invited to become Editor to the first
series of its quarterly journal, ‘The Ibis. Volume I appeared in 1859, and
the first series was completed in 1865. The next six volumes were edited by
Prof. A. Newton, and the third series by Osbert Salvin. From 1877 Sclater
again became Editor, either alone or in company with a partner, till the end
of the ninth series in 1912, and during this time he contributed many valuable
papers to the Journal. In 1908, on the occasion of the Jubilee, Sclater,
together with the three other surviving founders, F. D. Godman (President),
W. H. Hudleston, and P. S. Godman, received the gold medal of the Society.
With the British Association for the Advancement of Science he had a
long connection, and attended many of the meetings after he became a
member in 1847, including the visit to Montreal in 1884 and South Africa
in 1905, For several years he was Secretary of Section D, and at the
Bristol meeting in 1875 was its President, and delivered an address on
“The State of our Knowledge of Zoological Geography,” a subject which had
hitherto been much neglected. In geography he took a special interest ; he
became a life member of the Geographical Society, and was a constant
Philip Lutley Sclater. Vv
attendant at its meetings. He resigned the Secretaryship of the Zoological
Society in 1902 after forty-three years’ tenure of that office, and retired to
his country house, Odiham Priory, in Hampshire, but was still a frequent
visitor at both the Natural History Museum and the Library of the
Zoological Society till shortly before his death. He continued a constant
attendant at the dinners of the British Ornithologists’ Club, at which he
usually presided. At the last meeting, held on June 11, 1913, he was
presented by the club with an address, signed by nearly all the members, and
a piece of plate, in recognition of his services during the past twenty-one
years, but he was, unfortunately, too unwell to be present, as he was suffering
from a carriage accident, from the effects of which he died on June 27.
Sclater married in 1862 Jane Anne Eliza, youngest daughter of Sir David
Hunter-Blair, Bart., of Blairquhan, Ayrshire, and leaves a widow and three
sons and one daughter.
With a view to obtain collections of natural history, Sclater assisted in
promoting researches in foreign parts. Amongst these may specially be
mentioned Sir H. H. Johnston’s expedition to Kilimanjaro, Prof. Balfour’s
visit to Socotra, and many others. Sclater likewise travelled in many parts
of Europe and North America, visiting the museums, and making the
acquaintance of the principal zoologists.
As before mentioned, he commenced his collection of birds while an
undergraduate at Oxford, at that time intending to include those from all
parts of the world, but afterwards resolved to confine himself to Central and
South America alone, limiting himself to the orders Passeres, Picarie, and
Psittaci. This collection, containing 8824 specimens, representing 3158
species, including many types, was ultimately acquired by the Natural
History Museum.
Sclater received the honorary degree of Doctor of Philosophy from the
University of Bonn in 1860, and was made a Doctor of Science by the
University of Oxford in 1901. He was elected a Fellow of the Royal
Society in 1861, and served twice on the Council, was likewise a Fellow of
the Linnean, Geographical, and Geological Societies, and a member of
several other scientific societies both at home and abroad.
Amongst the works published by Sclater may specially be mentioned
‘A Monograph on the Tanagrine Genus Calliste, ‘ Zoological Sketches,’ by
J. Wolf, with notes by P. L. Sclater, ‘Exotic Ornithology, by P. L. Sclater
and Osbert Salvin, and the ‘Book of Antelopes,’ by P. L. Sclater and
Oldfield Thomas. In addition to these, he published over 1200 papers in
various periodicals, chiefly on birds and mammals, besides many others in
conjunction with Osbert Salvin, Forbes, and O. Thomas, etc. His last paper
in the ‘Ibis’ was issued in the January number, 1913, while his first in
the ‘ Zoologist’ in 1844.
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INDEX. to VOL. LXXXVIL.. (B)
After-images and successive contrast with pure colours (Porter and Edridge-Green), 190.
Anthocyan pigments of plants.—Part VI (Keeble, Armstrong and Jones), 113.
Anthocyanins and anthocyanidins, production of (Everest), 444.
Arber (KE. A. N.) On the Fossil Floras of the Wyre Forest, with Special Reference to
the Geology of the Coalfield, etc., 317.
Armstrong (HK. F.) See Keeble, Armstrong, and Jones.
Arterial pressure, measurement of (Hill and others), 344.
Avebury (Lord). Obituary Notice of, i.
Bacillus coli communis, decomposition of formates by (Grey), 461 ; decomposition
of glucose and mannitol by (Grey), 472.
Bacteria, oxidation of thiosulphate by (Lockett), 441.
Barratt (J.O. W.) The Nature of the Coagulant of the Venom of Hehis carinatus, a
Small Indian Viper, 177.
Bassett (A. Ll.) See Wheldale and Bassett.
Blacklock (B.) and Yorke (W.) The Trypanosomes causing Dourine (Mal de Coit or
Beschilseuche), 89.
Blood pressure, resonance of tissues in transmission of (Hill and others), 255.
Body weight and lethal dose of toxic substances (Dreyer and Walker), 319.
Brain, mid-, postural and non-postural activities of (Brown), 145.
Brodie (T. G.) A new Conception of the Glomerular Function, 571 ; and
Mackenzie (J. J.) On Changes in the Glomeruli and Tubules of the Kidney
accompanying Activity, 593.
Broom (R.) The Origin of Mammals, 87.
Brown (LT. G.) On the Question of Fractional Activity (“ All or None” Phenomenon)
in Mammalian Reflex Phenomena, 132; —On Postural and Non-postural
Activities of the Mid-Brain, 145.
Bruce (Sir D.) and others. Trypanosome Diseases of Domestic Animals in Nyasaland.
II1.—T. pecorum, 1; Morphology of Various Strains of the Trypanosome causing
Disease in Man in Nyasaland.—The Mzimba Strain, 26 ; —— The Trypanosome
causing Disease in Man in Nyasaland.—Susceptibility of Animals to the Human
Strain, 35; Plasmodium cephalophi (sp. nov.), 45; —— Trypanosomes of the
Domestic Animals in Nyasaland. I.—Z. simie, sp.nov. Part IJ.—The Suscepti-
bility of Various Animals to 7. s¢miw, 48; Part III, 58; ——- The Trypanosome
causing Disease in Man in Nyasaland. Part III].—Development in Glossina
morsituns, 516 ; Description of a Strain of Trypanosoma brucei from Zululand.
Part I.—Morphology, 493 ; Part 1I1.—Susceptibility of Animals, 511; Part I1T.—
Development in Glossina morsitans, 526.
Bullock (W. E.) and Cramer (W.) Contributions to the Biochemistry of Growth.—On
the Lipoids of Transplantable Tumours of the Mouse and the Rat, 236.
action
Chlorophyll extracts, formaldehyde as oxidation product of (Warner), 378 ;
of light on (Wager), 386.
Chloroplasts of green cells, presence of iron compounds in (Moore), 556.
Cholesterol content of growing chickens under different diets (Gardner and Lander),
229.
VOL. LXXXVII.—B. C
Vill
Church (A, H.) On the Floral Mechanism of Welwitschia mirabilis (Hooker), 354.
“Clot” formations, investigations on phenomena of, II (Schryver), 366.
Compton (A.) The Optimum Temperature of Salicin Hydrolysis by Enzyme Action
is Independent of the Concentrations of Substrate and Enzyme, 245.
Cramer (W.) See Bullock (W. E.) and Cramer (W.)
Creatine, excretion in carbohydrate starvation (Graham and Poulton), 205.
Croonian Lecture (Broom), 87 ; (Brodie), 571.
Darwin (Sir F.) On a Method of Studying Transpiration, 269 ; —— The Effect of
Light on the Transpiration of Leaves, 281.
Dosage of drugs (Dreyer and Walker), 319.
Dourine, trypanosomes causing (Blacklock and Yorke), 89.
Dreyer (G.) and Walker (EK. W. A.) The Determination of the Minimal Lethal Dose
of Various Toxic Substances and its Relationship to the Body Weight in Warm-
blooded Animals, etc., 319.
Dye (D. W.) See Glazebrook and Dye.
Echis carinatus, the coagulant of yenom of (Barratt), 177.
Edridge-Green (F. W.) See Porter and Edridge-Green.
Enzymes in decomposition of glucose, etc., by B. coli communis (Grey), 472.
Everest (A. E.) The Production of Anthocyanins and Anthocyanidins, 444.
Flack (M.) See Hill, McQueen, and Flack.
Flower-colour, chemical interpretation of Mendelian factors for (Wheldale and Bassett),
300.
Formaldehyde as oxidation product of chlorophyll extracts (Warner), 378 ; ——
synthesis of, from carbon dioxide and water (Moore and Webster), 163.
Fossil floras of the Wyre Forest (Arber), 317.
Gardner (J. A.) and Lander (P. E.) The Origin and Destiny of Cholesterol in the
Animal Organism. Part XI.—The Cholesterol Content of Growing Chickens under
Different Diets, 229.
Gel, formation of, from cholate solutions (Schryver), 366.
Genetics of tetraploid plants in Primula (Gregory), 484.
Glazebrook (R. T.) and Dye (D. W.) On the Heat Production associated with Muscular
Work, 311.
Graham (G.) and Poulton (E. P.) The Alleged Excretion of Creatine in Carbohydrate
Starvation, 205.
Gregory (R. P.) On the Genetics of Tetraploid Plants in Primula sinensis, 484.
Grey (E. C.) The Decomposition of Formates by Bacillus coli communis, 461 ; The
Enzymes which are concerned in the Decomposition of Glucose and Mannitol by
B. coli communis, 472.
Growth, biochemistry of (Bullock and Cramer), 236.
Gunda ulve, regeneration in (Lloyd), 355.
Gunn (J. A.) The Action of Certain Drugs on the Isolated Human Uterus, 551.
Hamerton (A. E.) See Bruce (Sir D.) and others.
Hammond (J.) and Marshall (F. H. A.) The Functional Correlation between the
Ovaries, Uterus, and Mammary Glands in the Rabbit, with Observations on the
(Hstrous Cycle, 422.
Helix pomatia, spermatocyte NAM of (Meek), 192.
Heredity in sea-urchins, studies in (MacBride), 240.
(bx
Hill (L.) and McQueen (J. M.) and Ingram (W. W.) The Resonance of the Tissues as
a Factor in the Transmission of the Pulse and in Blood Pressure, 255 ;
and Flack (M.) The Conduction of the Pulse Wave and the Measurement of
Arterial Pressure, 344.
Ingram (W. W.) See Hill, McQueen, and Ingram.
Jones (W.N.) See Keeble, Armstrong, and Jones.
Keeble (F.), Armstrong (E. F.), and Jones (W. N.) The Formation of the Anthocyan
Pigments of Plants.—Part VI, 113.
Kennedy (R.) Experiments on the Restoration of Paralysed Muscles by means of
Nerve Anastomosis. Part IJ.—Anastomosis of the Nerves Supplying Limb
Muscles, 331.
Kent (A. F. S.) Neuro-muscular Structures in the Heart, 198.
Kidd (¥F.) The Controlling Influence of Carbon Dioxide in the Maturation, Dormancy,
and Germination of Seeds.— Part I, 408 ; Part II, 609.
Kidney, glomerular function (Brodie), 571; changes in glomeruli and tubules
(Brodie and Mackenzie), 593.
Lander (P. HE.) See Gardner and Lander.
Life, origin of, photo-synthesis and, iron compounds in green-cell chloroplasts in relation
to (Moore), 556.
Lipoids of transplantable tumours (Bullock and Cramer), 236.
Lloyd (D. J.) The Influence of the Position of the Cut upon Regeneration in Gunda
ulve, 355.
Lockett (W. T.) Oxidation of Thiosulphate by Certain Bacteria in Pure Culture, 441.
MacBride (E. W.) Studies in Heredity. 11.— Further Experiments in Crossing British
Species of Sea-urchins, 240.
Macdonald (J. S.) Studies in the Heat-Production associated with Muscular Work, 96.
Mackenzie (J. J.) See Brodie and Mackenzie.
McQueen (J.) See Hill and others.
Malaria parasite of man, new (Stephens), 375.
Malarial parasites, growth, &c., in culture tube and human host (Thomson), 77.
Mammals, origin of (Broom), 87.
Marshall (F. H. A.) See Hammond and Marshall.
Medullosa pusilla (Scott), 221.
Meek (C. F. U.) The Ratio between Spindle Lengths in the Spermatocyte Metaphases
of Helix pomatia, 192.
Moore (B.) The Presence of Inorganic Iron Compounds in the Chloroplasts of the
Green Cells of Plants, considered in Relationship to Natural Photo-synthesis and
the Origin of Life, 556 ; and Webster (T. A.) Synthesis by Sunlight in
Relationship to the Origin of Life.—Synthesis of Formaldehyde from Carbon
Dioxide and Water by Inorganic Colloids, 163.
Muscular work, heat production associated with (Macdonald), 96; —— (Glazebrook
and Dye), 311.
Nerve anastomosis and restoration of paralysed muscles (Kennedy), 331.
Obituary Notices :—
Avebury, Lord, i.
Sclater, P. L., iii.
(Estrous cycle, observations on (Hammond and Marshall), 422.
x
Origin of life, synthesis by sunlight in relation to (Moore and Webster), 163.
Ovaries, uterus, and mammary glands, functional correlation between (Hammond and
Marshall), 422.
Pixell (H. L. M.) Notes on Toxoplasma gondit, 67.
Plasmodium cephalophi, sp. nov. (Bruce and others), 45.
Porter (A. W.) and Edridge-Green (F. W.) Negative After-Images and Successive
Contrast with Pure Spectral Colours, 190.
Poulton (E. P.) See Graham and Poulton.
Primula sinensis, genetics of tetraploid plants in (Gregory), 484.
Pulse, resonance of tissues in transmission of (Hill and others), 255.
Pulse wave and measurement of arterial pressure (Hill and others), 344.
Reflex phenomena, question of fractional activity in (Brown), 132.
Salicin hydrolysis by enzyme action, optimum temperature of (Compton), 245.
Schryver (S. B.) Investigations dealing with the Phenomena of “Clot” Formations.
Part IIl.—The Formation of a Gel from Cholate Solutions, etc., 366.
Sclater (P. L.) Obituary Notice of, iii.
Scott (D. H.) On Medullosa pusilla, 221.
Sea-urchins, experiments in crossing (MacBride), 240.
Seeds, influence of carbon dioxide on maturation, etc. (Kidd), 408, 609.
Sex ratio in Mus rattus, variations in, associated with unusual adult female mortality
(White), 335.
Stephens (J. W. W.) A New Malaria Parasite of Man, 375.
Synthesis by sunlight and origin of life (Moore and Webster), 163.
Thiosulphate oxidised by bacteria (Lockett), 441.
Thomson (J. G. and D.) The Growth and Sporulation of the Benign and Malignant
Tertian Malaria] Parasites in the Culture Tube and in the Human Host, 77.
Tissue growth in autogenous and homogenous plasma (Walton), 452.
Toxic substances, minimal lethal dose of, and relationship to body weight (Dreyer and
Walker), 319.
Toxoplasma gondii (Pixell), 67.
Transpiration, method of studying (Darwin), 269 ; effect of light on (Darwin), 281.
Tristichaceze and Podostemacex, lack of adaptation in (Willis), 532.
Trypanosoma brucei, description of strain from Zululand (Bruce and others), 493, 511,
526.
Trypanosoma simi, susceptibility of various animals to (Bruce and others), 48 ;
development in Glossina (Bruce and others), 58.
Trypanosome causing disease in Man in Nyasaland.—The Mzimba strain (Bruce
and others), 26 ; susceptibility of animals to Human strain (Bruce and others), 35 ;
development in G. morsitans (Bruce and others), 516.
Trypanosome diseases of domestic animals in Nyasaland. IIIl.—Trypanosoma pecorum
(Bruce and others), 1.
Trypanosomes causing dourine (Blacklock and Yorke), 89.
Uterus, action of drugs on isolated human (Gunn), 551.
Venom of Echis carinatus, nature of coagulant of (Barratt), 177.
Wager (H.) The Action of Light on Chlorophyll, 386.
Walker (E. W. A.) See Dreyer and Walker.
Walton (A. J.). Variations in the Growth of Adult Mammalian Tissue in Autogenous
and Homogenous Plasma, 452.
X1
Warner (C. H.) Formaldehyde as an Oxidation Product of Chlorophyll Extracts, 378.
Watson (D. P.) See Bruce (Sir D.) and others.
Webster (T. A.) See Moore and Webster.
Welwitschia mirabilis, floral mechanism of (Church), 354.
Wheldale (M.) and Bassett (H. Ll.) The Chemical Interpretation of some Mendelian
Factors for Flower-Colour, 300.
White (F.N.) Variations in the Sex Ratio of Mus rattus associated with an Unusual
Mortality of Adult Females, 335.
Willis (J. C.) On the Lack of Adaptation in the Tristichaceze and Podostemacez, 532.
Wyre Forest, fossil floras of (Arber), 317.
Yorke, W. See Blacklock and Yorke.
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LIQUID NITROGEN AND HYDROGEN. I.—THE MEAN ATOMIC SPECIFIC HEATS AT 50°
ABSOLUTE OF THE ELEMENTS A PERIOD ONO va teary ATOMS See
By Prof. Sir JAMEs Dewar, F.R'S. . 158
THE THERMAL EFFECTS ‘PRODUCED BY HEATING AND COOLING
PALLADIUM IN HYDROGEN. By J. H. Anprew, M.Sc., and A. Hort, M.A., D.Sc. .. 170
SPECTROSCOPIC INVESTIGATIONS IN CONNECTION WITH THE
ACTIVE MODIFICATION OF NITROGEN. III.—_SPECTRA DEVELOPED BY THE acne
CHLORIDES OF SILICON AND TITANIUM. By W. Jevons, A.R.C.Sc., B.Sc. (Plate 9) .. 187
ON THE PASSAGE OF WAVES UO UNE SETS fo THIN
OPAQUE SCREENS. By Lorp Ray.ezicu, O.M., F.R.S 194
EXPERIMENTS ON THE TEMPERATURE COEFFICIENT OF Es KEW
COLLIMATOR MAGNET. By G.A. SHAKESPEAR, M,A., D.Sc. «4 220
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