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i.® 1 a
PROCEEDINGS OF
THE ROYAL SOCIETY.
Szcrion B.—BrotoagicaL ScrENcES.
The Effect of Plant Growth and of Manures upon the Retention
of Bases by the Soil.
By A. D. Haut, M.A., and N. H. J. Mritugr, Ph.D.
(Communicated by H. E. Armstrong, LL.D., Ph.D., F.R.S. (From the Lawes
Agricultural Trust.) Received March 30,—Read May 11, 1905.)
Introductory.
The following investigation deals with the changes in the amount of calcium
carbonate, the chief substance in the soil acting as a base, which are brought
about by natural agencies, by manuring, and particularly by the growth of
plants.
Since Cavendish discovered that calcium carbonate dissolves in water
charged with carbon dioxide, and ascertained the presence of calcium bicar-
bonate in many natural waters, it has been recognised that the calcium
carbonate present in most soils must be subject to regular loss. The air
enclosed in the soil contains a considerable proportion of carbon dioxide
derived from the roots of plants and decaying organic matter (Wollny, for
example, found amounts varying from 3 to 14 per cent. by volume), and the
. soil water, after attaining a state of equilibrium with the gas at this partial
pressure, becomes an effective solvent of any calcium carbonate that may be
present, removing it as bicarbonate into the drains or the general stock ot
underground water. That such dissolution does take place in the upper
layers of the soil is evident from the analyses made by A. Voelcker* and
* © Journ. Chem. Soc.,’ 1871, p. 276.
VOL, LXXVII.—B. B
2 Mr. A. D. Hall and Dr. N. H. J. Miller. —[ Mar. 30,
Frankland* of the drainage waters from the Broadbalk wheatfield at
Rothamsted.t Although the tile drains, from the flow of which the waters in
question are derived, rarely lie more than 2 feet 6 inches below the surface of
the soil, yet the drainage water from the unmanured plot contained on the
average 99 parts per million of CaO, of which 84 were in the state of
bicarbonate, and the water from the plot receiving farmyard manure every
year contained 147 parts of CaO per million, of which 72 may be regarded
as in the state of bicarbonate. Such concentrations, representing a hardness
of about 17 and 26 degrees respectively, though by no means equal to
those of truly calcareous waters, are above the average of natural waters
in this country; yet, as will be seen later, the calcium carbonate from
which they are derived is practically confined to the uppermost 9 inches
of soil.
While such losses may be regarded as natural, it has long been known
that many of the substances applied to the soil under the general term of
artificial manures react with the calcium carbonate there present and bring
about its dissolution. Liebig, for example, pointed out that the di-hydrogen
calcium phosphate (“superphosphate”) contained in bones made soluble by
treatment with sulphuric acid, reacts at once with the bases in the soil and
becomes again insoluble. The researches of Wayt and A. Voelcker§ showed
further that the retention of ammonium and potassium salts by cultivated soils
is always preceded by a double decomposition with calcium carbonate, the
bases being retained as carbonates while the acids appear in the drainage
waters combined with calcium. A. Voelcker’s analyses of the Rothamsted
drainage waters,|| when dealing with the plots receiving salts of ammonium,
potassium, etc., as manures, show the same reactions taking place on a large
scale.
Again, the process of nitrification, going on in all normal soils, requires
some base to combine with the nitrous and nitric acids produced by the
oxidation of the ammonia and other nitrogen compounds.§ In an ordinary
way this base is supplied by calcium carbonate, hence a further source of
loss to the calcium carbonate of cultivated soils.
The soils of the Rothamsted experimental plots afford peculiar facilities for
the study of the rate at which these losses of calcium carbonate, both natural
* “Journ. Roy. Agri. Soc.,’ 2nd Series, vol. 18, 1882, p. 14.
+ See Table X.
t ‘Journ. Roy. Agri. Soc.,’ 1st Series, vol. 11, 1850, p. 313, and vol. 13, 1852, p. 123.
§ ‘Journ. Roy. Agri. Soc.,’ 1st Series, vol. 21, 1860, p. 105, and vol. 25, 1864, p. 333.
|| Loc. cit.
{7 ‘Instruction sur létablissement des Nitriéres, Paris, 1777; Warington, ‘Trans.
Chem. Soc.,’ 1879, p. 429.
1905. ] On the Retention of Bases by- the Soil. 3
and induced by the application of saline manures, are taking place. In most
cases the plots have received the same mauures, year after year, for more
than fifty years, and though unfortunately samples of the soil were not taken
at the starting of the experiments, yet in the case of the Broadbalk Field,
on which wheat has been grown continuously since 1843, a set of samples
drawn in 1856 has been preserved, in addition to samples drawn in 1865 and
in 1893. From the Hoos Field (continuous barley since 1852) samples were
drawn in 1868, 1882, and 1904—5, and from Agdel]l Field (under a four-
course rotation since 1848) samples exist which were drawn in 1867, 1874,
1883, and 1905.
Furthermore, the calcium carbonate in the Rothamsted soil is of
extraneous origin, and is entirely localised in the surface layer which is
stirred by the plough. The subsoil, from which the surface soil is
undoubtedly derived, belongs to the drift formation of “ clay-with-flints,”
characteristic of the chalk plateau, and consists of the débris of the chalk
formation largely mixed with sands and clays of the Reading series.* It
normally contains little or no calcium carbonate, although it is partly
derived from the chalk formation and rests upon the solid chalk at a
varying depth of 8 to 12 or 20 feet.
In the eighteenth century, however, a characteristic feature of the
agriculture of this district of Hertfordshire was to manure the land by sinking
pits through the clay to the chalk, which was then lifted and spread in
considerable quantities. Arthur Youngf quotes from Walker’s Survey of
1795—* the now prevailing practice of sinking pits for the purpose of
chalking the surrounding land therefrom . . . The most experienced Hert-
fordshire farmers agree that chalking of lands so circumstanced is the best
mode of culture they are capable of receiving.” Evidence of the former
prevalence of this practice of chalking may be seen by the existence in
each of the Rothamsted fields of a “dell,” a depression representing the
fallen-in pit from which the chalk was extracted. A certain rawness of the
soil round the edges of these “dells” still bears witness to the disturbance
ereated by the excavation, though it is known that nothing of the kind was
done during the late Sir John Lawes’ possession of the estate, which dates
back to 1834. Probably the pits were but little worked after the close of the
eighteenth century, and certainly neither chalk nor lime has been applied to
the plots since they were put under experiment. At the present time the
* See H. B. Woodward, ‘“‘Report of the Soils and Subsoils of the Rothamsted Estate.
Summary of Progress of the Geological Survey, 1903.”
+ “‘ Report on the Present State of the Agricuiture of Hertfordshire,” presented to the
Board of Agriculture, 1804,
B 2
4 Mr, A. D, Hall and Dr. N. H. J. Miller. [Mar. 30,
chalk is visible only in the upper soil, and is there present in small rounded
nodules varying in diameter from 3 or 4 mm. downwards.
I.—CALCIUM CARBONATE IN ROTHAMSTED SOILS.
A. Analytical,
The first section of this paper deals with the determinations of the amounts
of calcium carbonate present in the soils and subsoils of certain of the plots
in the Broadbalk, Hoos, Agdell, and Little Hoos Fields, the samples having
been drawn at the dates specified above and again in 1904—5. As the
calcium carbonate is of artificial origin, and was probably distributed with
considerable irregularity, however much this may have been equalised by
the subsequent working to which the soil has been subjected, it cannot be
expected that the samples analysed will represent the whole soil of the plots
with the same degree of accuracy as would be attained in the case of some
original constituent of the soil.
Arable soils only are considered ; with soils in permanent grass the question
is complicated by the well-known action of earthworms, which, as demon-
strated by Darwin, bury the surface layer by constantly bringing fine subsoil
to the top.
The determinations of calcium carbonate have been made by means of an
apparatus described by one of us in conjunction with Dr. E. J. Russell.*
The results are calculated from the volume of carbon dioxide evolved on
treating the fine soil with dilute sulphuric acid in vacuo, due provision being
made to bring into account the carbon dioxide remaining dissolved in the
reacting liquid. In a few cases, where the percentage of calcium carbonate
was very low, the carbon dioxide evolved by treating a considerable quantity
of the soil with acid was absorbed by caustic soda and determined by double
titration. For these determinations we have to thank Mr. Arthur Amos,
B.A. The amount of magnesium carbonate present is too small to affect the
results, and in any case, as the real quantity sought is the amount of readily
available base in the soil, it is desirable to express it always in the same terms.
The soil samples were all taken in the same way: a steel frame 6 inches or
1 foot square and 9 inches deep is driven into the ground, and its contents are
carefully picked out; this gives the soil proper. The surrounding soil is
then dug away and the frame is driven down another 9 inches. The contents
now represent the subsoil at the second depth of 10 to18 inches. The process
is then repeated to as many holes as may be required. In this way samples
are taken from four, six, or eight holes on each plot, according to its size.
* Hall and Russell, ‘Trans. Chem. Soc.,’ vol. 81, 1902, p. 145.
1905. | On the Retention of Bases by the Soil. 5
The samples, after drying at a temperature not exceeding 60° C., are roughly
powdered and put through a woven wire sieve with a mesh of } inch (this
sieve passes a little more than the 3 mm. round-hole sieve now commonly
used). From the fine earth thus obtained from each hole composite samples
representing the whole plot at each depth are then made up, of which portions
are finely ground for analysis.
Table I gives details of the manurial treatment of the various plots in
Broadbalk and Hoos Fields, in the soil of which the calcium carbonate has
Table 1—Nature and Quantities per Acre of the Manures annually applied.*
aa Ba || a $ g 3 6 | 86
eee] EE s|aelee| 4 | 4) 22] | 43
ae BS ge] sa | Se ze| ea | ge] os
3 2h 8 ag cess) |p Slew) EE) etsy | ee per ster
S BOF aa Se \se | ea) & | 4/83) 32)/ a3
cr Sie ey Sa cet ON AV racy Macey taal fot coal rosy neta fer= tak
|
Broadbalk Field.
tons. lbs. lbs lbs. lbs ewts lbs lbs lbs
Sls dadaseneces 1844 | Unmanured |’ — — — _— = = =
Bee Acca: 1844 14 — — — — — — = =
Mgcounoonanee 1852 — — — — — 83 955 200 100 100
(qhatoonnenees 1852 — — 100 100 — 3°5 200 100 100
Wausae ecco ts 1852 — —_— 200 200 — 3°5 200 100 100
Sieichaeeecks 1852 — — 300 300 — 3°35 200 100 100
Ora rtaet sacri 1852 — 275+ — — — 3°5 200 100 100
ALO vrrehctne siciessers 1845 — — 200 200 — = = = —
IE atone 1849 —_— — 200 200 — 3°5 [ices = =
| i
Hoos Field.
NOG aire 1852 Unmanured | — — — — —_ — = —
BOW asda 1852 — = — — — 3°5 200 100 100
ALEKS lanterns 1852 — 100 100 — = = — =
AA ey cv 1852 —_ — 100 100 | — 3°5 200 100 100
Neyer oe 1868 — 275 — _— — = = =
ANG ee sirsoeine 1868 — 275 — — — 3°5 200 100 100
IG e barra: 1852 — — — — 1000 -—— — = =
(ee 1852 14 — — — — = — = =
if
* For certain minor variations in the amounts of manure applied see “ Memoranda of the Field
and other Experiments at Rothamsted. Lawes Agricultural Trust, 1901.”
ft 550 lbs. per annum up to 1884.
been determined; Table II gives the average weight in pounds per acre of the
fine dry soil in the layer 9 inches deep which is removed by the sampling
tool for the different fields at each of the specified dates; Table III for
Broadbalk, Table V for Hoos, and Table VII for Agdell and Little Hoos Fields
give the percentages of calcium carbonate in the fine soils dried at 100° C.
Mr. A. D. Hall and Dr. N. H. J. Miller.
[Mar. 30,
Certain difficulties are experienced in attempting to calculate from these
figures the actual quantity of calcium carbonate per acre in the soil at different
periods, owing to the impossibility of drawing samples that represent the same
layer of soil on each occasion.
Since changes of texture are set up by the
different treatment of the plots the consolidation of the surface layer varies,
so that the 9-inch slice includes more or less soil from time to time.
On
most plots the weights of the samples tend to get heavier, because the soil
sets more closely together under the conditions of long-continued manuring
Table Il—Weights of Fine Soil Dried at 100° C. per Acre.
Broadbalk Field.
Plots. Depth. 1856. 1865. 1881. 1893. 1904.
lbs. lbs. lbs. lbs. lbs.
OE ee Ne eh 1st 9 inches =! 2,200,000 | 2,400,000 | 2,400,000 | 2,400,000
Other plots ......... 1st 9 inches } 2,200,000 | 2,300,000 | 2,560,000 | 2,650,000 | 2,650,000
JA plots eee 2nd 9 inches | 2,590,000 | 2,590,000 | 2,590,000 | 2,590,000 | 2,590,000
All plots .....0..0.. 3rd 9 inches | 2,815,000 | 2,815,000 | 2,815,000 | 2,815,000 | 2,815,000
Hoos Field.
|
Plots. Depth. 1868. | 1882. 1904-5.
lbs. lbs. lbs.
PIR RASA Res, con to 1st 9 inches — 2,100,000 2,100,000.
Other plots ......... 1st 9 inches | 2,400,000 | 2,400,000 | 2,400,000
JNU FTIOES soccascoanec 2nd 9 inches 2,721,000 2.721,000 2,721,000
INT FIO poncosseooce 3rd 9 inches | 2,891,000 | 2,891,000 | 2,891,000
Agdell Field.
Plots. Depth. | 1867. | 1874. 1883-4. 1905.
lbs. lbs. lbs. lbs.
IND Faleta Gessoonedoce 1st 9 inches 2,140,000 | 2,400,000 | 2,500,000 | 2,500,000
INTIUFSGEG Soocopposnee 2nd 9 inches 2,450,000 | 2,450,000 | 2,450,900 | 2,450,000
Little Hoos Field.
Plots. Depth. 1873. | 1904.
lbs. lbs.
All plots ............ 1st 9 inches 2,500,000 2,500,000
All plots ............ 2nd 9 inches 2,500,000 2,500,000
1905. | On the Retention of Bases by the Soil. id
Table I1l—Broadbalk Wheat Soils—collected at various dates.
Calcium Carbonate per cent. in Fine Soil Dried at 100° C.
Keel
: Sept., | Oct., | Oct., | Oct., | Sept.,
nk Manures. | 1856. | 1865. | 1881. | 1893. | 1904.
1st Depth (1—9 inches).
|
| (D> |} YG | je 1 p. ¢
26 | Farmyard manure (14 toms) ...............eeeeee eee [== 4-20 7) 3°79 | 3-46 | 3°28
& || Wharrnensiny200ll Cosqauoosconnbeccectos beoeseees Soe ocseecnarers | 5°35 | 4:54 | 3:97 | 3°45 | 3°29
Smee Ullerminerals, se. deacssselseswiainrsctrica seacencnsswcets 5°65 | 4°96 | 3°75 | 3°34 | 2:94
6 a +200 lbs. ammonium salts ......... ; — | — 3°41 1°98 | 2°33
7 5D + 400 Ibs. mi | 8 noceandes | — | 3-82 | 3:19 | 2°36 | 2-25
8 x +600 lbs. OLR Sh eee & een en oEea elite 7o0 lot 76
9 i + 275* lbs. nitrate soda ............ | A249) 3-99 9 Se72)" || 3-36
10 | 400 lbs. ammonium salts only ................0000 | 5-41 | 4-10 | 3:31 | 2°76 | 2-47
11 55 0 and SHPO PEN. o| 4°36 | 3:14 | 2°76 _—
i } | |
2nd Depth (10—18 inches).
2b | Farmyard manure (14 tons) ............00s0eseeees — | 0°277 | 0°310 | 0-422 | 0-237
& | havering cacosenobeqoos ono Goddoo Loe oso pEdade nna pedese — | 0°222 | 0-162 | 0:099 | 0:116
Ome eualemnin eras)” hectic ects cite otaku sisenedgercue senses — | 0:147 | 0-128 | 0:100 | 0-110
6 Fs +200 lbs. ammonium salts ......... } =e | = 0:110 | 0:114 | 0-132
fl 5 +400 Ibs. a, Decne esses — | 0:212 | 0:210 | 0:096 | 0-167
8 a +600 lbs. ih, BEAN Be p= — | 0-106 | 0-093 | 0-117
9 3 + 275* lbs. nitrate soda ............ | — | 0:3809 | 0:263 | 0:482 | 0°143
10 | 400 lbs. ammonium salts only ..............0..040. | — | 0-127 | 0:187 | 0:170 | 0°111
11 e Fe and superphosphate.... — | 0:119 | 0:179 | 0 107 | —
3rd Depth (19—27 inches).
|
| 26 | Farmyard manure (14 tons) ..............00 ee — | 0°181 | 0°121 | 0°130 | 0-095
fo Ge AML ney G8 206 Le soode der cao Bas ana mdocnbd BeleSeneeUnd aANOEOnES — |0°179 | 0:090 | 0-084 | 0-113
Ope pEiallimineralsy Woecnes as scelee sic sesce eneseie sencaseceee | — | 0-056 | 0:079 | 0:050 | 0-100
6 * +200 lbs. ammonium salts ......... } — — 0-058 | 0:073 | 0:112
7 ss +400 Ibs. te AER a, | — | 0-144] 0-115 | 0-075 | 0-136
8 és +600 lbs. he Pay aA eee — | 0-063 | 0-104 | 0-122
9 55 + 275* lbs. nitrate soda ............ — | 0:144 | 0:102 | 0:130 | 0-116
10 | 400 lbs. ammonium salts only ...................5 — | 0:073 | 0-070 | 0-070 | 0°105
11 5 3 and superphosphate...) — | 0-090 | 0-089 | 0°085 ==
* Double this amount applied from 1855 to 1884 inclusive.
with saline manures and the gradual loss of organic matter. On the contrary,
the soil of the plot receiving farmyard manure grows lighter through the great
accumulation of organic matter. These changes of weight would be of little
moment were the soil uniform, but as the calcium carbonate is almost wholly
present in the surface soil down to about six or seven inches, small variations
in the thickness of the slice taken by the tool cause a varying admixture of
the poorer subsoil, and so may induce considerable change in the estimated
8 Mr. A. D. Hall and Dr. N. H. J. Miller. [Mar. 30,
weights of calcium carbonate. It is impossible to eliminate wholly the errors
thus introduced, especially in the case of Broadbalk, where the average weights
of the 1865 samples are exceptionally low and where the subsoil shows also
a good deal of variation.
In Table IV two estimates have been drawn up for the amounts of calcium
carbonate per acre. In the upper set of figures an attempt has been made
Table [V.—Broadbalk Soils.
Calcium Carbonate in Lbs. per Acre.
Rate of loss per acre
per annum.
Plot. 1865. 1881. 1893. 1904.
Whole | 1881—1904.
period.
Total of Ist and 2nd 9 Inches+a fraction of the 8rd 9 Inches, to bring them
all to the same Total of 5,240,000 Lbs. of Soil per Acre.
lbs. lbs. lbs. lbs. Ibs. Ibs.
QD icceconiscs 100,400 99,300 94,300 85,100 392 618
Big eaee ap 110,800 105,900 94,000 90,200 528 683
55. seSaoaon0%04 118,100 99,400 91,100 80,800 809* 809
(\ Rostpogaconee — 90,200 55,400 65,200 1086 1086
Uaciiejecsinnaien' 93,900 87,200 65,000 64,000 767 1009
Geer tee = 75,500 48,300 49,700 1122 1122
Qe vawisctescnse 106,000 109,100 111,100 92,700 341 713
OY isonsgcoon06e 97,800 88,400 77,500 68,300 756 874
Tete 103,700 85,100 75,900 = 993 =
First 9 Inches only, reckoned as weighing 2,500,000 Lbs.
ZDesaeecsewace 105,000 94,700 86,500 82,000 590 552
33 ‘esondaosb0ar 113,500 99,200 86,200 82,200 800 739
SEA 124,000 93,700 83,500 73,500 878* 878
Gas: reds = 85,200 49,500 58,200 1174 1174
Ms saecad sys 95,500 79,750 59,000 56,200 1010 1024
Buss Boe Bs 71,000 43,200 44,000 1174 1174
Oe rainiejs cece 106,000 99,700 93,000 84,000 564 683
OM rear sicaee se 102,500 82,700 69,000 61,700 1045 913
a ea 109,000 78,500 69,000 a 1429 =
* 1881—1904 only.
to introduce a correction for the varying thickness of the slice by adding to
the weights of calcium carbonate in the first and second depths such a pro-
portion of the third depth as would ensure the comparison of an equal weicht
of soil in all cases. The lower set of figures is based simply upon the per-
centage of calcium carbonate in the upper layer of soil, assuming a general
1905.]| On the Retention of Bases by the Soil. 9
average weight of 2,500,000 lbs. for the fine dry earth in the top 9 inches of
soil, thus leaving out of account both the changing weights of the upper slice
and the contents of the subsoils. The two sets of figures lead to much the
same comparative results, but the lower table is to be preferred as free
from any speculative corrections, bearing in mind, however, that the rate of
loss is probably over-estimated on most of the plots and under-estimated
on the plot receiving farmyard manure. Fig. 1 shows these percentages of
calcium carbonate in the upper soil plotted against the time.
Fic. 1.—Percentage of Calcium Carbonate. Broadbalk Field. First 9 inches.
A cursory examination of the figures and curves shows that if it may be
assumed that the calcium carbonate was equally distributed over the whole
field initially, then the greatest losses have occurred on the plots manured
with ammonium salts, the loss increasing with each addition of ammonium
salts; sodium nitrate, on the contrary, would seem to have exercised some
preservative influence on the calcium carbonate, which is now at its maximum
on the plot where that manure is used.
Table VI gives the weights of calcium carbonate per acre in soils from the
Hoos field as calculated from the percentages in Table V and from the average
weights of soil in Table II, without any attempt at correction ; the results
being also thrown into a graphic form in fig. 2.
10 Mr. A. D. Hall and Dr. N. H. J. Miller. [Mar. 30,
Table V.—Hoos Field Barley Soils, collected at various dates.
Calcium Carbonate per cent. in Fine Soil dried at 100° C.
Plot. Manures. 1868. 1882. 1904-5.
1st Depth (1—9 inches).
Per cent. Per cent. Per cent.
nos aareacace Winimian Ure ercavavswoencaentoeseorenck netic moneee — 3°43 2 34
416) Gacwadoda Mall tnin erall’soiaccovanwece pounce scecsw asc teers 1:78 1°47 ©) 7/1
DA eserey sees 200 Ibs. ammonium salts ...........cc0eeeeeee ees —_ 3°26 2°53
ATKU ERE Cs Full minerals + 200 lbs. ammonium salts ... 1°34 0°87 0°28
AEN fasteners 275 Ibs. nitrate of soda ..........ccsescseeeeeseees — 3°30 2°59
AINienrenee Full minerals + 275 lbs. nitrate of soda ...... 1-30 1°32 0-48
ot eeerpoade 100 Tbs:\rape cakerecs..ssce-neeeevesesaseseaesen: — 1°58 0°89
ees 14 tons farmyard manure ...............06.e.000 — 1°92 1:'14
2nd Depth (10—18 inches).
Gy eragsas Mnmanured encrcdcaasceeosctee sees See — 0-107 0 -202
MOwennstoac: Hall sminerallsh eanscccsostees cas nena eee ee meas 0°110 0-091 0:139
MUAY eter ce 200 Ibs. ammonium salts .............c.0seeeeeee — 0-116 0°119
AN Neer meat Full minerals + 200 lbs. ammonium salts ... 0-154 0-074 0-083
TENT aves 275 lbs. nitrate Of Soda ........500..esseescensn ess — 0°107 0-112
AN ceneesnen | Full minerals + 275 lbs. nitrate of soda ...... 0-064 0-073 0-112
MO osoens000 1000 Ibs. rape cake .........0.......0scccceseneee ens — 0-095 0 066
7T—2 14 tons farmyard manure ................2.60000+ = 0-275 0-147
3rd Depth (19—27 inches).
MOU setaee LUhatre te hahvnys\s engnnnser seacododosen nbabcasedonoocabocsc6 — 0 :056 0-078
AO: sites Full minerals -asrcscannsosaerearaeinnirnen eee 0 :094 0-081 0-091
AAS estes 200 Ibs. ammonium salts ..............00e0 eee ees —_ 0-063 0 062
ATAGD Sexsratrcts Full minerals + 200 lbs. ammonium salts ... 0 096 0-090 0:073
TEN Ragodaane 275 lbs. nitrate of soda .............0ceeceeeee eee — 0-075 0-084
ANE een Full minerals + 275 lbs. nitrate of soda ...... 0 ‘063 0-061 0 °103
I) aeopese0 NOOO Ms srapel Cake cwawscreccis sdecadeeeseceeeeeeches — 0 068 0 067
72 w, 14 tons farmyard manure ............ce0eeeeee eee — 0-074: 0-103
Unfortunately the figures show at once that the initial chalking of the
Hoos Field has been very irregular, the plots numbered 1 show more
than twice as much calcium carbonate as the corresponding plots numbered 4.
As also in this field, samples only exist for two dates, 1882 and 1904—5,
instead of the four which are available for the Broadbalk Field, less weight
can be laid on the results for the individual plots. It is, however, noticeable
that it is one of the plots receiving ammonium salts every year which now
contains the least calcium carbonate.
1905. | On the Retention of Bases by the Soil.
Table VI.—Hoos Field Soils,
Calewwm Carbonate in Lbs. per Acre.
Rate of loss
Plot. 1868. 1882, 1904-5. per acre
per annum.
Total for Three Depths (27 inches).
Ibs. Ibs. lbs. lbs.
I: coossocosvs00o0 == 86,800 63,900 1000
Aone) Seen 48,431 40,100 23,400 675
AY Goo podedooboeHd = 83,200 65,700 760
4, a0 cb8000 000000 39,100 25,500 9,900 790
TL a ae es 84,300 67,600 725
Csi dial 34,754 35,400 17,500 G5
16 ke eae pas 42,500 25,100 755
eRe scisinaycasiane — 49,900 30,900 830
First 9 inches only, reckoned as weighing 2,500,000 lbs.
110) -sonesostoanatos = 85,750 58,500 1185
AR eh Aa 44,500 36,750 17,750. 723
SG AR a syateslseisistasie = 81,500 63,250 793
Lyk scala 33,500 21,750 5,750 750
IGN! soosassooneaden = 82,500 64,750 772
Zi en ae 32,500 33,000 12,000 554
NOW as tjcesort = 39,500 22,250 750
(22 ae aa 48,000 28,500 848
80000 lbs,
IN
IA
pa0)
60000 lbs.
1868
Fic. 2,—Calcium Carbonate, lbs. per acre.
1882
1905
Hoos Field. To 27 inches.
11
12 Mr. A. D. Hall and Dr. N. H. J. Miller. [Mar. 30,
Table VII gives the percentages of calcium carbonate found in the soils
of the Agdell and the Little Hoos Fields, which are not specifically
manured.
Table VII.—Calcium Carbonate per cent. in Fine Soil dried at 100° C.
Agdell Field.
| 1867. | 1874. | 1883-4. | 1905.
|
1st Depth (1—9 inches).
Unmanured ... 5 815 5 984 5 102 4, 522
Unmanured ... 5 912 6 °414 4,927 4-567
2nd Depth (10—18 inches).
Unmanured ... 0 °304 0-514 0-137 0-185
Unmanured ... 0 220 0 “767 0°134 07138
Little Hoos Field.
1873. 1904. |
1st 9 inches... 3-974 2-704
2nd 9 inches... 0 °145 0:118
Lastly in Table VIII the rates of loss in all cases are brought together for
comparison. In calculating these rates of loss the earliest’ samples (1856)
from the Broadbalk Field have been rejected as not comparable with the
Table VIII.—Rate of Loss of Calcium Carbonate from Soil, lbs. per Acre
per Annum.
Broadbalk| Hoos Agdell | Little Hoos,
wheat. | barley. | rotation. various.
Wmmannuredeereacpeeenesers cere Cereeeereere cree 800 1000 { eae 1046
Mineral manures only...............s.sccssssseeeeee 880 675
- +200 lbs. ammonium salts ... 1170 790
bs +400 Ibs. i eel ORO =
bs +600 lbs. ‘ eel LO =
+ 275 lbs. sodium nitrate ...... 565 465
200 lbs. ammonium salts only ... — 760
400 lbs. ) 6 40 1045 _—
275 lbs. sodium nitrate only ... — 725
Farmyard manure ................+. 590 830
Ra pekcakenscsnc cus sormacte swe secession cwatacieptecteetecne — 755
1905. | On the Retention of Bases by the Soul. 13
others, the rate of loss is then based upon the difference between the earliest
and latest samples from each plot.
B. Loss of Caleiwm Carbonate on Unmanured Land.
It will be convenient to consider the unmanured plots together, so as to get
an idea of the initial loss of calcium carbonate to the soil when there are
no disturbing influences introduced by the manure. All the unmanured soils
show the same general characteristics—a comparatively large amount of
calcium carbonate in the top 9 inches, varying from nearly 6 per cent. on
Agdell Field down to little more than 2 per cent. on Hoos Field. The second
depth, however, contains as a rule between 0:1 and 0:2 per cent., only in a
few special cases does it rise above the latter figure; in the third depth the
proportion is 0-1 per cent. or less. As the Rothamsted fields are situated on
almost the highest levels of the chalk plateau, it is difficult to suggest any
reason for the restriction of the calcium carbonate to the surface soil other
than its artificial origin, and this conclusion is confirmed both by the irregu-
larity in the amount found in different fields and by its absence where the
land has not been under cultivation. Samples were taken from three places
on the adjoining Harpenden Common, where the same class of land occurs
at approximately the same level as the experimental fields, but which has
never been in cultivation and now carries a growth of poor grass, gorse, and
bracken.
The results :—
Soil of Harpenden Common, 1 to 9 inches deep... 0°210 p. ce. CaCOs.
s . 10 to 18 r coo OG a
show that initially the soil of the Rothamsted Estate must have been practi-
cally devoid of calcium carbonate.
The successive determinations of the amounts present in the soil of the
cultivated fields give the following rates of loss per acre per annum for
the unmanured land: Broadbalk 800 lbs.; Hoos, 1000 lbs.; Agdell, 922
and 938 lbs.; Little Hoos, 1046 lbs. or an average loss of over 944 lbs.
per acre per annum due to percolating water only. These rates are all
probably too high, because of the gradual exhaustion of the organic matter
and the resultant consolidation of the soil which has affected the later
samples. But even after making all corrections that are possible for this
source of error, the rate of loss would still amount to 800 lbs. per acre.
These estimates may be to some extent checked by certain determinations
made in 1896—8 of the amount of calcium compounds present in the water
percolating through the drain gauges at Rothamsted. These drain gauges are
14 Mr. A. D. Hall and Dr. N. H. J. Miller. [ Mar. 30,
blocks of undisturbed soil of 1/1000 acre in area, isolated by impervious walls
from the surrounding land and maintained without vegetation since 1870.
Table IX shows the proportion of lime in the water during 20 months
Table [X.—Lime in Drainage Water percolating through 60 inches of Soil,
September, 1896, to April, 1898.
Drainage through | CaO in drainage
Rainfall. soil, 60 inches water
deep. per million.
: inches. inches.
1896.
September............... 8-077 6 °362 60-1
Octobereerr grees 4,°132 2 992 56 °3
November............... 1 °387 0 833 46 -1
December ............... 4-416 3 811 49 °8
1897.
JANUary .....eeeeeeeees 2-031 1 585 48 -9
February ............... 2-925 3 °264 47 °9
4-197 2 589 48 -2
1-913 0 °320 48 °4;
1-718 0 :047 53 °3
2 °734 0 865 541
0:467 0 -024 44) °5
3-238 0:105 61°0
2-440 0872 60 °5 ]
0-960 0-001 _—
1-048 0-110 50 *4
3 503 3 060 56 °6
1898.
January ...........---- 0-795 0 821 53 ‘1
February ............... 1-098 0-047 30°35
Marci tte 1-060 0: 492 51 °5
AoA, saqnooceoons 00800008 1 443 0 -082 43 °2
Average ......... 2-479 1-414 53 °5
(September, 1896, to April, 1898); the average concentration is 53°5 parts of
CaO per million for the gauge with 60 inches of soil, which on the average
percolation of 13:8 inches would give an annual loss of calcium carbonate of
300 lbs. Two causes contribute to make this figure low. In the first place the
soil of the gauges is not very rich in calcium carbonate, determinations made on
samples taken in 1870 from the land immediately adjoining gave 3:06 per
cent., while two small samples bored out from the actual gauges in 1905 gave
1:88 per cent. for the upper 9 inches. Secondly, the air contained in the
soil of this plot must be comparatively deficient in the carbon dioxide
1905. | On the Retention of Bases by the Soul. 15
necessary to bring the chalk into solution; as a rule the soil gases get richer
in carbon dioxide the greater the depth, but the soil of the gauge is cut off
from the subsoil and open to the atmosphere at the 60-inch depth. The long
absence of any crop or manure will have reduced to very small limits the
amount both of organic matter decaying to carbon dioxide, and the organic
sulphur compounds which by bacterial action become sulphuric acid and leave
the soil as calcium sulphate. These causes will co-operate to lessen the
removal of calcium carbonate from the soil in the gauge, and as a matter of
fact the concentration of 53°5 parts of CaO per million observed in its
drainage is only about half of the concentration of the water running from
the tile-drains beneath the unmanured plot in Broadbalk, which according to
Voelcker’s and Frankland’s analyses (Table X) amounted in the mean to
about 99 parts per million. But assuming that this latter figure represents
the average proportion of lime in the drainage water from the unmanured
plot, and that the average annual percolation through the soil of this plot is
10 inches,* equal to that through the 60-inch gauge, the annual loss of calcium
carbonate per acre should amount to 400 Ibs. for the unmanured plot instead
of the 800 lbs. found by analysis of the soil of the plot. The number of
analyses, however, upon which the former estimate is based, is too small for
great accuracy.
Table X.—Broadbalk Drainage Water.
Mean of 10 analyses by Voelcker and Frankland. Parts per million.
Total solid | Lime and
uO LEAS. matter. magnesia.
2 Farmyard manure (14 tons) ..............5 367 -2 123
G) gravel 4! |) Whaoneb mb oyerel sco sdocessogoodvaGaud ade coonoonnoees 227 °8 99
5 Mineral shonllygueaesceseesesasecrccccsccnoaas 329 °8 132
6 » +200 ]bs. ammonium salts ...| 450°3 171
7 » +400 Ibs. . yl eee| B42 “4 207
8 » +600 Ibs. a Sed GBS 222
9 » +275 lbs. nitrate of soda ...... 405 °7 126
10 400 Ibs. ammonium salts alone ............ 441 ‘8 173
11 is 4 +superphosphate...| 490 °4 197
In a paper by Creydt, von Seelhorst, and Wilmst on the composition of
the drainage waters from an ordinary field tile-drained at a depth of
* Ten inches was the estimate formed by Lawes, Gilbert and Warington, ‘Journ.
Roy. Agri. Soc.,’ 1882, vol. 48, p. 24; Warington, ‘Trans. Highland and Agri. Soc.,’
1905, vol. 17, 5th series, p. 168, estimates the drainage as somewhat more than 8-2 inches,
while a comparison between the concentration of chlorine in the water from the unmanured
plot and from the 60-inch drain gauge would lead to an estimated drainage of 9 inches.
+ ‘Journ. der Landw.,’ 1901, p. 251.
16 Mr. A. D. Hall and Dr. N. H. J. Miller. [Mar. 30,
53 inches, the annual loss of lime is estimated at 630 kilogrammes per
hectare, almost exactly equivalent to 1000 lbs. of calcium carbonate per
acre. Unfortunately, the proportion of calcium carbonate in the soil is not
given, but the agreement with our figures for the removal of calcium ecar-
bonate is very satisfactory. |
Another consequence of some interest follows from these determinations
of the loss of calcium carbonate from the unmanured plots. The analyses
already quoted of the drainage water from the unmanured plot of Broadbalk
Field show about 100 parts of lime per million, equivalent to a loss of about
400 lbs. of calcium carbonate per acre in the surface soil instead of
800 Ibs. estimated from the analysis of the soil. But the deep-seated waters
of the chalk contain on the average about 150 parts of lime per million, a
deep well at Harpenden, for example, yielding 158 parts per million.*
Assuming that the percolation through the unmanured plot on Broadbalk
represents the average percolation over the chalk area, then the removal of
chalk by solution would be in the ratio of the concentrations of the two waters
in question, 2.c., the average annual denudation of the chalk by solution alone
would amount on the one estimate to 600 lbs., or on the other to 1200 lbs.
per acre per annum. As the specific gravity of the chalk is about 2-2, and
it contains from 95 to 99 per cent. of calcium carbonate, this would mean a
lowering of the surface by solution alone at the rate of either 1/11000 or
1/5500 of a foot per annum. These estimates depend upon the assumption
that the percolation through this unmanured plot of arable land represents
the percolation over the whole area of the chalk, whereas ordinary crops or
even grass would cause increased transpiration and allow of less percolation.
But on the contrary, the figure adopted for the concentration of the lime in
the drainage water from the unmanured plot is based on only a small number
of analyses and would appear to be too low. The concentration of the
drainage waters would be increased by any use of organic manures, by heavy
cropping or by permanent vegetation, all of which would increase the pro-
duction of the carbon dioxide causing dissolution in the soil. For example
the analyses of the water from the plot receiving farmyard manure every
year show an average of 123 parts of lime per million instead of 99 from the
unmanured plot. This increased proportion of lime in the water percolating
through ordinary land may be set off to some extent against the lessened
percolation due to crops, but on the whole the evidence is in favour of the
lower rate, so that a denudation of about 1/10000 foot per annum is a more
probable figure.
The accuracy of the sampling is not sufficient to enable any conclusion to be
* Warington, ‘Trans. Chem. Soc.,’ vol. 51, 1887, p. 543.
1905. | On the Retention of Bases by the Soil. 17
drawn as to whether the loss of calcium carbonate fluctuates with the rainfall
and percolation during each period. But the magnitude of the annual loss is
somewhat surprising; assuming it to be only 800 lbs. per acre, then the
Broadbalk Field must have contained at least 70 tons per acre of chalk at the
beginning of the nineteenth century, and still contains so much that it will
not be exhausted by the end of the present century. As, also, the rate of
loss will probably fall with each reduction in the quantity present, dissolution
being proportional to the surface exposed, the period of complete exhaustion
will be considerably postponed. Since much of the value of the land agricul-
turally, both in its fertility and in its ease of working, depends on the
presence of calcium carbonate, it is clear that for the last 100 years the
agricultural community have in this respect been living upon the capital
accumulated by their forefathers, and are taking no steps to replace the
inevitable depletion of this capital.
C. Effect of Manures upon the loss of Calcium Carbonate.
Most of the manured plots under investigation receive a dressing of
“mineral manures” in addition to the varying amounts and compounds of
nitrogen. This mineral manure consists of 34 cwt. per acre of superphosphate
containing 17 per cent. of soluble phosphoric acid (equivalent to 37 per cent.
of calcium phosphate “made soluble ”), 200 lbs. of potassium sulphate, and
100 lbs. each of magnesium and sodium sulphates. Of these substances the
superphosphate reacts immediately with the calcium carbonate of the soil, the
sparingly-soluble di-calcium hydrogen phosphate being precipitated wherever
the superphosphate solution comes in contact with a particle of chalk in the
soil in accordance with the equation
CaH,P203 + CaCO3 = Ca2H2P20s3+ CO2+ H20.
To complete this reaction, the 34 cwt. of superphosphate would require
about 47 lbs. of calcium carbonate, but so small an annual loss would hardly
be perceptible in the analyses.
The neutral sulphates of potassium, sodium, and magnesium should occasion
no loss, for though they react with calcium carbonate, the resultant alkaline
carbonate is retained by the soil and would be estimated as calcium carbonate
by the method of analysis adopted, which is based upon the carbon-dioxide
evolved on treating the soil with acid. The action of the plant also,
discussed later in this paper, would probably result in the reconversion of the
sodium and potassium carbonates into calcium carbonate. The action of the
mineral manures, as seen in the analyses of the soil, has not occasioned
sufficient loss of calcium carbonate to be apparent within the limits of accuracy
VOL. LXXVII.—B. Cc
18 Mr. A. D. Hall and Dr. N. H. J. Miller. [ Mar. 30,
of the determinations. In the Broadbalk Field, Plot 10 can be compared with
Plot 7; both receive the same amount of ammonium salts, but Plot 7 receives
the minerals in addition. The rate of loss is practically identical, 1010 lbs. per
acre per annum on Plot 7 and 1045 lbs. on Plot 10. Unfortunately Plot 5,
receiving minerals only without nitrogen, cannot be compared with the
unmanured Plot 3, because both Plots 5 and 6 show an entirely exceptional
rate of loss, not to be accounted for unless it be that one of the dells, from
which the field was originally chalked, lies in these two plots, and may have
caused a very irregular distribution of the chalk. But if the rate of loss on
Plot 5 be calculated over the period 1881—1904 only, it amounts to 880 lbs.
per acre against 800 lbs. per acre on the unmanured plot.
In the Hoos Field the results are too irregular to bear much discussion,
for the plots which receive minerals—4o, 4a, 4N—start with less than half
the chalk contained by the corresponding 1o, 1a, and 1N.
In the plots, however, which receive ammonium salts as a manure, the loss
of calcium carbonate is much increased. A reaction of the type
(NH4)2SO4 + CaCO; => (NH4)2CO3 + CaSO.
takes place as soon as the ammonium salts are dissolved, the ammonium
carbonate is adsorbed by the surface action of the humus and the finer clay
particles of the soil until it is nitrified, while the calcium sulphate passes
forthwith into the drainage water. When heavy rain follows the application of
the ammonium salts to the Broadbalk wheatfield, only traces of ammonia find
their way into the drains, whereas there is an immediate great increase in
the calcium sulphate and chloride present in the drainage water. At
Rothamsted the manure termed ammonium salts consists of an equal mixture
of ammonium sulphate and chloride, and is applied to the various plots at the
rate of 200, 400, and 600 lbs. per acre, quantities which would react with
161, 321, and 482 Ibs. respectively of calcium carbonate. Before, how-
ever, the ammonium carbonate thus produced has been long in the soil a
second molecule of calcium carbonate must be consumed to provide a base for
the nitrous and nitric acids formed by its nitrification. This would make
the loss caused by the application of 200 lbs. of ammonium salts up to
321 lbs. of calcium carbonate, half of which is caused by the initial reaction
producing ammonium carbonate, and half by the nitrification of the latter.
In order to determine the rate of loss in the field, it will be convenient, in
the various cases where a comparison is possible, to subtract the average
rate of loss on a plot without ammonium salts from the plots receiving
ammonium salts, and divide by 2 or 3, as need be, to find the effect in each
case of 200 lbs. of ammonium salts.
1905. | On the Retention of Bases by the Soul. ug
Annual loss of calcium
carbonate.
Broadbalk (Plot 7—Plot 3)+2......... 105 lbs. per acre.
i (Plot 10—Plot 3)+2......... 122 3
3 (Plot 8—Plot 3)+3......... 125 Ss
Hoos (Plot 44—Plot 40)+ 1 ...... 115 rs
Mean...... iiy/ 7"
This mean value for the loss caused by 200 lbs. of ammonium salts is not
very far from the 161 lbs. estimated above as necessary to convert them into
ammonium carbonate, but neither the mean nor any of the individual
analyses support the view that a second molecule of calcium carbonate is
removed from the land by the nitrification of the ammonium carbonate.
However, this nitrification must take place; indeed, there is every evidence
that it takes place so rapidly and thoroughly in the Rothamsted soil that no
ammonium salts are carried forward un-nitrified from one season to the next.
The analyses of the drainage waters (Table X), while they show a progressive
increase in the amount of lime for each addition of ammonium salts in the
manure, do not permit any estimate to be formed of the rate of removal, so
much is the extent of the percolation, as seen in the relative frequency with
which the drains run, affected by the size of the crop, which becomes large on
the heavily-manured plots. It remains, therefore, to be explained why the
loss of the soil should be at the rate of one rather than of two molecules of
calcium carbonate for every two molecules of combined ammonia applied in
the manure.
The plots receiving sodium nitrate in place of ammonium salts show not
only no special loss of calcium carbonate due to the nitrogeneous manure, but
a distinctly diminished loss as compared with the unmanured plot. On
Broadbalk the nitrate plot loses at the rate of 564 lbs. per acre against
800 lbs. on the unmanured plot; on Hoos the loss is 465 lbs., against 679 lbs.
on the plot receiving the same minerals but no nitrate.
That the sodium nitrate exercises some conserving influence on the calcium
carbonate in the soil is also apparent from a consideration of the analyses of
the subsoil. On Broadbalk, where the subsoil from 10 to 18 inches contains
in most cases 0:1 or 0:15 per cent. of calcium carbonate, the same layer
beneath the plot receiving sodium nitrate shows 0°31, 0:26, 048, and
0:14 per cent. at the various dates, while the third depth of this plot is also
richer than on the corresponding plots.
Further evidence may be derived from the composition of the drainage
waters (Table X); the water from the plot receiving mineral manures only
Cc 2
20 Mr. A. D. Hall and Dr. N. H. J. Miller. [Mar. 30,
contains 132 parts of lime per million, the plot receiving the same minerals
and sodium nitrate gives a drainage water containing 126 parts of lime per
million. Thus where the nitrate is used there is both a lower concentration
of lime in the drainage water and a smaller total percolation, because of the
much greater crop, and consequently increased transpiration cn this plot.
The sodium nitrate then either saves the calcium carbonate of the soil from
its normal loss or has some power to bring about the re-formation of calcium
carbonate. With this fact must be correlated the non-disappearance from
the other plots of the calcium carbonate required to form calcium nitrate with
the nitrified ammonia base.
Considering lastly the plots receiving farmyard manure, the Broadbalk
Field shows a much lower rate of loss on the plot manured every year in
this way than on the unmanured plot, 590 lbs. against 800 lbs. per acre per
annum. The corresponding plots in the Hoos Field hardly confirm this
view, since both the plot receiving farmyard manure and that receiving rape
cake, the only other organic manure employed, appear to be losing calcium
carbonate at much the same rate as all the other plots. However, as the
Hoos Field results rest upon determinations made at two dates only instead
of four as in the case of Broadbalk, it is much more probable that the result
yielded by the latter is trustworthy. The subsoil of the plots receiving farm-
yard manure also show amounts of calcium carbonate above the normal—0:28,
0°31, 0:42, and 0:24 in Broadbalk, and 0:28 and 0:15 in Hoos Field. The
drain beneath the farmyard manure plot on the Broadbalk Field runs but
rarely, because the humus derived from the long-continued organic manuring
of this plot is capable of temporarily absorbing any ordinary rainfall and then
passing it gradually down to the subsoil without causing the drain to run.
But the few analyses that have been made of the water draining from this
plot indicate a lower concentration in calcium compounds than would be
expected from the large amount of carbon dioxide produced by the decay of
recent organic matter, and also from the considerable annual addition of
calcium compounds in the manure itself. The composition of farmyard
manure is very variable, but the mean of a number of analyses gives 0°6 per
cent. of CaO, or an annual application of 190 lbs. per acre, of which the
greater part is in combination with organic acids. There is, therefore, an
addition of calcium compounds in the manure more than equivalent to their
greater concentration in the drainage water, and the net result is a gain of
calcium carbonate to the soil as shown by the diminished rate of loss on this
as compared with the unmanured plot.
We thus obtain three lines of evidence that there is some agency saving or
re-creating the calcium carbonate of the soil: (1) the loss of calcium carbonate
1905. | On the Retention of Bases by the Soul. PAL
induced by the use of ammonium salts is less than half that required for the
absorption and subsequent nitrification of the ammonia; (2) where sodium
nitrate or (3) where farmyard manure is applied, the rate of loss of calcium
carbonate is below that of unmanured land.
Further evidence that there must be under normal conditions some action
at work protecting or renewing the bases of the soil may be gathered trom
the continued fertility of many soils containing but a trace of calcium
carbonate. The following analyses may be quoted of soils that have fallen
under the personal observation of one of us, soils which, despite their very
low content of calcium carbonate, have continued to give crops under arable
cultivation for a long period.
Table XI.—Calcium Carbonate per cent. in various Soils.
Pomanton: London j London | Gault| Weald | Bagshot | Thanet Woburn Experimental Farm,
clay. clay. | clay. | clay. sand. beds. Stackyard Field. Barley.
Tocalit Wanboro’,| Ashtead | Alder Sen Bisl Woodnes- 1876 1908. | 1908. 1903.
y: Surrey. | Common. | Holt. pager aaa borough. ‘| Plot 2a.| Plot 3.| Rotation.
p- ¢. p- ¢. 1b @ || je p- ¢. p. ¢. p. ¢. 2G: b @ p. ¢.
Ist depth ...| 0:°065 0:002 | 0:04 | 0-037 | 0-008 0-018 0:087| 0-051 | 0-070] 0-089
2nd depth...| 0-084 nil 0:16 | 0-012 | 0-016 0-010 0 066 044. 042 | 0-071
Another striking case is afforded by the Stackyard Field on the farm of the
Royal Agricultural Society at Woburn, which has been under experiment
since 1876. Table XI also shows a series of determinations of calcium
carbonate in the soil of this field taken at the beginning of the experiments
and in 1902. The amount of calcium carbonate present is exceedingly small,
barely determinable in fact, yet the plots continue to yield normal crops,
except those which have been manured with ammonium salts. The latter
in recent years have become almost sterile, showing an acid reaction to litmus
paper and refusing to grow wheat or barley unless they first receive a dressing
of lime.*
Now in all these cases, however low the proportion of calcium carbonate
may be, the action of the percolating water must remove some of it, and the
recurring process of nitrification also demands a base. Yet the small quantity
of base available does not disappear entirely so as to render the soil unfertile,
unless some specially calcium carbonate consuming material, like the
ammonium salts, is employed asa manure. The continued fertility of such
* See J. A. Voelcker, ‘Journ. Roy. Agri. Soc.,’ 3rd Series, vol. 10, 1899, p. 585, and
vol. 62, 1901, p. 272.
22 Mr. A. D. Hall and Dr. N. H. J. Miller. = [ Mar. 30,
soils almost devoid of calcium carbonate has long been a problem, but it
now seems probable that the calcium carbonate and other bases which are
required for nitrification are in some way returned to the soil as bases, and
that when a ready-formed nitrate like sodium nitrate is used as a manure
there is an addition of available base to the soil or a corresponding diminu-
tion in the amount of calcium carbonate removed by the drainage water.
Furthermore these or other agencies conservative of calcium carbonate are
sufficient to maintain the quantity in the soil at the level for comparatively
healthy growth. Of the possible conservative actions, two will be now con-
sidered and evidence be brought to show that (1) the normal growth of plants
leaves behind a residue of base in the soil, (2) the decay of plant tissues
results in the production of calcium carbonate.
II.—Errect or PLANT GROWTH ON THE REACTION OF THE SOIL.
The plant, it is well known, does not take up the salts of the soil water in
the proportions in which they are present in the solution, but exercises a
selective action in favour of substances necessary to the nutrition processes,
such as potash and phosphoric acid. And if the composition of the ashes
of the plant be taken into account, it is clear that the selective action is
exercised not merely on the salts with which the root is in contact but on
their acids and bases considered separately. For example, from a solution of
calcium nitrate the plant would withdraw more nitric acid than its equivalent
of lime and from a solution of potassium sulphate more potash than its
equivalent of sulphuric acid.
When a plant is burnt the ash is usually alkaline, because the organic acids
and any nitrogen present as nitrate in the plant are all driven off, leaving the
bases as carbonates. But when a balance is struck between the acids and
bases in the ash and when the nitrogen present in the plant before burning is
calculated as an acid, since it all entered the plant as nitrate, the acids are
generally to be found in excess. Warington,* indeed, has already pointed out
with reference to the published analyses, that plants must retain more acids
than bases. It does not appear to have been noticed, however, that such a
result, by leaving behind in the soil a corresponding excess of base, must
have an appreciable effect upon the reaction of the soil, although Knop and
other early investigators have observed that the solutions in which plants
are grown as water cultures become alkaline after a time. It is hardly
possible to decide whether the excess of base is left behind in the soil water
* “ Agricultural Students’ Gazette,’ 1899, p. 133 ; see Lawes and Gilbert, ‘Journ. Roy.
Agri. Soc.,’ vol. 55, 1894, p. 640.
1905. | On the Retention of Bases by the Soul. 23
or excreted by the root cells. The nutrient constituents pass through the cell
wall of the root hairs by osmosis until the concentration is the same on
either side ; such substances as are required by the plant are then withdrawn
from action by the protoplasm, thus lowering the concentration on the inside
and causing a fresh diffusion of that particular substance through the cell
wall. Looking at the question from the standpoint of the ionic hypothesis,
the soil water would be a highly ionised solution, in which any given ion will
pass by osmosis into the root-hairs as long as the plant maintains the tension
of that ion lower on the inside of the cell than in the solution outside. As
the plant is always transforming the nitrogen, sulphur and phosphorous from
the condition of inorganic acids in which they enter into neutral or even
basic organic compounds, and since it also as a rule utilises more of these
substances- than of the metallic bases, such acid ions will be withdrawn by
the protoplasm in greater quantity, and so must pass through the cell wall
from the external solution at a greater rate than the corresponding basic ions,
the necessary equilibrium being maintained by the carbonic acid excreted by
the root cells.* From this point of view, when the root is drawing nutriment
from a solution of a neutral nitrate, the nitric acid ions would be travelling
inwards to the protoplasm and the carbonic acid ions outwards, so that the
carbonate of the base might be considered as forming outside the cell wall.
If, on the other hand, the salt be considered to move into the cell
undissociated and there to lose its nitric acid to the protoplasm, the base must
then be supposed to diffuse out again as carbonate. The net result, however,
is the same under either hypothesis, viz., that after the plant has been
growing for some time in a neutral solution it will have taken up an excess
of acid and left a corresponding excess of base, now combined as carbonate,
in the solution representing the water of the soil.
The following Table (XII) shows the composition of several crops as per-
centages and again recalculated as equivalents of hydrogen, phosphoric acid
being reckoned as tribasic, since the soil solution will be mainly derived from
tribasic phosphates in the soil.
It will be seen that there is a considerable excess of acid in the plant, from
which it follows that an equivalent amount of base was left behind in the
soil. This base is in most cases nearly equivalent to the nitrogen taken in as
nitrate, and calculated as calcium carbonate will amount to between 100 and
300 lbs. of calcium carbonate per acre. In other words the normal growth of
farm crops leaves behind from the salts in the soil used for its nutrition about
as much base as would have been previously required for the nitrification of
* Wor a discussion of the electrical disturbances such an interchange would involve, see
Kohn, ‘ Landw. Versuchs.,’ vol. 52, 1899, p. 315.
24 Mr. A. D. Hall and Dr. N. H. J. Miller. — [ Mar. 30,
Table XII.—Basic and Acid Constituents of various Crops.
Agdell Agdell Agdell
rotation. rotation. rotation. Panne
Wheat. Barley. Swedes. ys
Complete Complete Complete oe
1856-1873.
manure. manure. manure.
§ courses. 8 courses. 3 courses.
Dry matter per acre...............0.5 A749 4124 3899 4812
Nitrogen per cent. in dry matter... 0°88 1-00 2°41 1°55
Ash per cent. in dry matter......... 4°74, 4:00 6°59 7 +24
Percentage Composition of Ash.
iHerricloxid eWeceesse-ceeseeseessinns ess 0°37 0 64 0-96 1°32
IRWIN®. Soooo0boocc00Ds090G0Ng20 cap noAOBAGOO 4-41 7 82 13 *42 8°27
IMaemesiay face op-cessensesennceaces: 2°98 4°16 2°70 3 42
JEOUERAO, — Scrodoooaadoscsoosangnce000b0c009 16°21 19 33 36 °33 35 °59
DOA mehr cissulee sasaiseems cose att telomeres 0°39 1°67 4-27 3 87
Phosphoric acid .........-..s.+e000 9-09 12 -88 9-38 7:96
Sulphuric acid ..................cc000 2°56 3°58 12 -36 5°76
(Cli erStA® soonsecoaococos nao gaac0Nbaq000000 1°46 3°15 4°18 14°83
SHINER, Sacnadcoosndsscoc0 dco 00000b990000000 60 °58 46°47 1 04 16°54
Constituents reduced to Equivalents of Hydrogen, and Lbs. per Acre.
TaleraaV OPAC) Goaccosebsoo sob nocdon500860 0-03 0 04 0-09 0°17
Wik EM eewcwnetecss be ceswissaisitelsevinaelecslece 0°36 0-46 1°24 1-03
MB OMESIA ae .iiseseteneceiste sceeednccese | @©88 0°34 0°35 0-60
IPOtas henna sneeweccneenacts je sceties ce cies 0°78 0°68 a99 2°64
S Oda sessintns deesseueneroes Scuecmeasrs 0:03 0-09 0°36 0 -44,
Basesstotaly Seocccrsnacceceecsecsiere 1°53 1°61 4°03 4°88
Phosphoric acid ...............-.-++» 0°87 0:90 1-02 1°18
Sulphuric acid .............0c..eseeeee 0:14 0-15 0-80 0°50
@hlorine ee sccceccersncoscictcteecestece 0:09 0°15 0-30 1-47
INTEV OREN aectiristun ceiesioseesuescemereenues 3:00 2°97 6°74 5°35
Sillcar- iva woescaanasacsOenaceeessessesent 4°53 2°55 0:09 1°91
Acids, total, excluding silica ... 4°10 4:17 8 86 8 50
MotaltotibAasest esc tecste eee 1°53 1°61 4:03 4°88
Excess of acid (as hydrogen) ... 2°57 2°56 4°83 3°62
Equivalent to calcium carbonate 129 1238 242 181
the nitrates which entered the plant, as measured by the nitrogen finally
contained in it.
Although the many ash-analyses which have been made of farm crops
afford conclusive evidence of this restoration of base to the soil, it seemed
desirable to submit the fact to experimental verification. In 1903 water
cultures were made of barley and cabbage in normal solutions which were
1905. | On the Retention of Bases by the Soil. 25
contained in cylinders of Jena glass holding about 3 litres; it was found that
after growth had gone on for some time the solutions had gained in alkalinity
by an amount equivalent to the nitrogen taken up by the plant, less the
alkalinity of the plant ash. One example of these trials will suffice.
A cabbage plant made 7525 grammes dry matter containing 2°705 per cent.
of nitrogen = 02035 gramme nitrogen, equivalent to 0:01454 gramme
hydrogen. The ash of the plant had an alkalinity equivalent to
0701082 gramme hydrogen. The culture solution gained alkalinity
= 0:00558 gramme hydrogen, which with the 001082 gramme alkalinity of
the ash makes 0:0164 gramme of alkaline hydrogen found in ash and solution,
to correspond with the 0°01454 gramme of acid hydrogen equivalent to the
nitrogen in the plant.
In 1904 six cultures were started with wheat in similar jars on March 3,
and growth was continued without changing the solutions until June 11,
when the grain was fully formed. Three grains were sown in each jar and
growth was extremely vigorous, but was continued a little too long, for at the
close of the experiment no nitrate remained in the solution. For purposes of
analysis a mixture was made of Nos. 1 and 2, and of 3 and 4, the remaining
two being kept in reserve. Table XII{ shows the results obtained.
In the initial solution there was a trifling excess of acid over base owing
to the presence of a little ferric chloride, of which the acid alone is brought
into the account. At the end it will be seen that there is an apparent gain
of nitrogen, due in the main to dust (the greater part being present in the
sediment which formed in each jar) and the seed (which contained
Table XIII—Wheat Grown in Water Culture. Composition of Plant and
Solution after Growth.
Original Solution.
|
| Quantities | Equivalent to
| taken. hydrogen.
| | grammes. gramme.
Tee eta | 42190 0 -0898
IWEO) coseacoonosapoaeoeee 0 6018 0 -0301
(ORK) ceccaccsapenbenendeereee | 28000 0 -1000
Total bases | = 0 -2199
N (as nitrate)............| 1 -4000 0 -1000
P.O; (monobasic) ...... 2 0974 0 -0295
SiG eine A Be eee 1 -2080 0 -0302
WO pears tuscchs astaecttnsees 21658 0 -0608
Total acid......| _— 0 2205
26 Mr. A. D. Hall and Dr. N. H. J. Miller. [Mar. 30,
TABLE XIl1—continued.
After Growth. Nos. 1 and 2. Dry Matter produced = 93°7 Grammes.
Quantities found. Equivalent to hydrogen.
‘ : ieee ;
Sediment. | Solution. Plant. Sediment. | Solution. Plant. Total.
LoS ae ed alles zee aoeea| Boe hl SS oe es sid
gramme. | grammes. | grammes. || gramme. | gramme. | gramme. | gramme.
LENO) Fath aone — 0 5014 3 6644 —_— 0 01065 0 :07796 0 ‘08861
MgO ...... (0 -2082*) 0 -2318 0-1618 (0 01041) 0 01159 0 -00809 0 -08009
CORK Opascsneon 0-152 1 -1592 1 ‘4371 0 :005438 0 :04140 0 :05140 0 :09823
0 -01584 0 -06864 0 °13745 0 21693
ING eto 0 °1249 0 0103 1 °3540 0 :00892 0 00074 0 :09670 0 ‘106386
1240 Pe eoesoer 0 °2942 0 °5631 1 246 0 -00414 0 :00793 0 :01755 0 :02962
SOs ede — 0 6448 0 -4008 — 0 01612 0 :01002 0 :02614
0) ieeaa goaacnd — 0 °8114 1 °3461 — 0 -02285 0 03792 0 06077
0-01306 | 0-04764 | 0-16219 | 0-22289 |
Bases im excess...........+ +0 :00278 |+0 -01600 |=0 02474 |—0 °00596
After Growth. Nos. 3 and 4. Dry Matter produced = 77-6 Grammes.
KG Oe. sees —_— 0 -6736 3 4819 — 0 01483 0 07410 0 08843 |
MgO ...... (0 °1296*)| 0:°2413 0 :2809 (0 :00648)| 0 -01206 0 -01154 0 -03008 |
CaO sav occks 0 °2240 1 +2222 1 °3414 0 :00800 0 04365 0 :04790 0 -09955
0 01448 0 :07004 0 183854 0 -21806
WAN Wiiscesmsseee 0 :0972 0 :0080 1 4088 0 :00694 0 00055 0 10027 0 °10776 |
1B Oa Baotop 0 °345 0 °5765 1:170 0 :00486 0 -00812 0 -01648 0 :02946
SOnnn — 0 ‘6610 - 0 °3959 — 0 -01652 0 -00990 0 :02642
CCl lbeeseen coos —_— 0 8595 1 °2212 — 0 -02421 0 08440 0 ‘05861
| 0 :01180 0 :04940 0 °16105 0 22225 |
Bases in excess......-..... +0 00268 | +0 02064 |—0:02751 |—0-00419 |
* Estimated figure—these two determinations were lost.
00036 gramme nitrogen, equivalent to 0:00026 gramme hydrogen), and partly
to a trace of ammonia in the large quantities of distilled water evaporated
during growth. This excess of nitrogen accounts for the slight excess of acid
over base in the final result when all the nitrogen is reckoned as acid. It
will be seen that the solution and sediment (representing the soil) gained in
the one case base equivalent to 0:01878 gramme of hydrogen and in the other
to 0:02332 gramme of hydrogen, quantities which would probably have been
increased had the growth not been continued until all the nitrate was
exhausted.
It has been often supposed that plants excrete some organic acid from the
1905. | On the Retention of Bases by the Soil. 27
root which aids in bringing about the solution of nutrient materials in the
soil,* but no evidence could be found that anything except carbon dioxide
passed from the roots into the culture solution. After growth a considerable
bulk of the culture solution was evaporated to dryness and gently heated,
very slight signs of charring were observed, no more than could be attributed
to the dust, and the residue showed the same alkalinity before and after
ignition, as would not be the case had any organic acid excreted from the
root been present. On one occasion freshly precipitated ferric hydrate was
introduced into the solution as a source of iron; although this was distributed
all over the young growing roots so that it could not be shaken off, the plants
began to suffer from lack of iron, and continued to do so until a trace of
ferric chloride was introduced. Despite the well known acidity of the root-
sap there seems no evidence that in normal cases it ever passes outside the
cell wall, as long as the roots are unbroken.
These experiments then afford experimental justification for looking to the
growth of the plant as an explanation of some of the difficulties raised by the
rate of loss of calcium carbonate on the different plots. The analyses of
crops already quoted serve to show that the return of base to the soil may be
large, quite sufficient to make up for the calcium carbonate required each
year for nitrification. Hence soils which start with very small proportions of
calcium carbonate may yet preserve their healthy condition and permit of
nitrification, the losses caused by which are thus repaired.
Again it becomes intelligible that the use of ammonium salts as a manure
only occasions the loss of one molecule of calcium carbonate for each
two molecules of ammonia, since the second molecule required for nitrification
will be more or less restored during the growth of the plant. It has already
been shown that the actual loss of calcium carbonate to the soil caused by
the use of 200 lbs. of ammonium salts approximates to 161 lbs., and not the
322 lbs. which would be required if the calcium nitrate produced by nitrifica-
tion were wholly removed from the soil.
Further, when nitrate of soda is used as a manure, from the neutral
sclution in the soil of calcium or sodium nitrate an excess of acid will be
taken by the crop, leaving the soil richer in base. Hence the conservative
action of sodium nitrate on the calcium carbonate of the soil that is visible
in the analyses of both Broadbalk and Hoos Fields. It is possible to
calculate the amount of base restored to the plots receiving nitrate of soda
on the assumption that they possess the same average composition as
the wheat and barley in Table XII, and that the amount of base returned
* See Czapek, ‘ Pringsheim’s Jahr. f. wiss. Botanik,’ vol. 29, 1896, p. 321; Kossowitsch,
‘Ann, de la Science Agronomique,’ 2nd Series, vol. 1, 1903, p. 220.
28 Mr. A. D. Hall and Dr. N. H. J. Miller. [Mar. 30,
will be in proportion to the size of the crop. In this way the following
results are obtained :—
Table XIV.
Broadbalk Field. Hoos Field.
Plot 8. | Plot 9. | Plot 40.} Plot 4x.
lbs. Ibs. lbs. lbs.
Total produce (grain and straw) ........ ...seescecsecenceweee 1936 6133 2343 5524
Bases restored to the soil as calcium carbonate, cal- )
culated from total produce ..........-.--.cecsccssecereen senses 53 167 73 ayal |
| Mean rate of loss of calcium carbonate from the soil ... 800 564 675 465
| ———
| Total annual consumption of calcium carbonate ...| 853 731 748 636
The agreement between the figures in the last line is not very close, but
indicates that the restoration of base to the soil, as calculated from the
increase of crop on the plots receiving nitrate of soda, is approximately
equivalent to the lower rate of loss of calcium carbonate found on analysis of
the soil of these plots.
The results, as a whole, go to show that the action of plants, in leaving
behind a basic residue from the neutral salts in the soil upon which they
feed, is a very essential feature in the chemistry of the soil, explaining,
amongst other things, the maintenance of healthy conditions on the many
soils poor in calcium carbonate. It also serves to explain one or two other
points which have been observed in connection with the use of sodium nitrate
as a manure. It has long been noticed that the continued use of sodium
nitrate is very destructive to the texture of a clay soil, intensifying all the
clay properties, rendering the soil persistently unworkable when wet, and
forming hard and intractable clods when dry. The ultimate cause of such an
effect is the “deflocculation” of the fine particles composing the soil; they
are no longer bound together in loose aggregates, but are separated so as to
give the soil its most finely grained character. Such deflocculation of the soil
can be brought about by a trace of any soluble alkali, just as the opposite state
of flocculation is induced by a slightly acid reaction. The Rothamsted soils
continuously manured with sodium nitrate show marked signs of defloccu-
lation, the drainage water from the nitrated plots in the Broadbalk Field is
always more turbid than that from the other plots, and as one of us has
shown,* there results in time on the nitrated plots a perceptible washing
* Hall, ‘Trans. Chem. Soc.,’ 1904, vol. 85, p. 964.
1905. | On the Retention of Bases by the Soul. 29
down of the finest particles set free by the deflocculation into the subsoil or
the drains. The bad texture of the soil following on the use of sodium nitrate
is particularly to be seen on the mangel field, where it reaches its maximum
on the plots receiving sodium nitrate and other neutral alkali salts like
potassium sulphate and sodium chloride: it has been repeatedly observed to
be at its worst in the winter and spring after a large crop has been grown on
the sodium nitrate plots. As the soii of this field contains but little calcium
carbonate, some of the base left behind in the soil by the growth of the crop
would consist of bicarbonate of sodium or potassium, especially where the
other alkali salts are applied in the manure, and there would be quite enough
free alkaline carbonate thus formed to cause a thorough deflocculation of the
soil. This explanation would agree with the observed fact that the defloccu-
lation is much diminished where superphosphate only, an acid manure, is used
in conjunction with the sodium nitrate.
IT.—Errect or OrGANIC MANURES ON THE REACTION OF THE SOIL.
Although the evidence is not so trustworthy as in the case of sodium
nitrate, yet the use of farmyard manure and of rape cake seems also to result
in a diminished rate of loss of calcium carbonate to the soil. Some of this
may be due to the lessened percolation consequent on the greater water-
retaining power of the soil enriched in humus, but another cause may be
sought in the bacterial decomposition of calcium salts in the organic débris.
Farmyard manure contains various calcium salts derived from the vegetable
matter out of which it has been formed, sometimes in their original form, but
partly broken down into the undefined carbon compounds known as
“humates.” Calcium humate, Wollny* has already shown, can be converted
into calcium carbonate by bacteria present in the soil, while the following
experiments show that the commonest of all calcium salts in the plant, the
widely distributed calcium oxalate, is readily fermented to carbonate.
100 c.c. of a nutrient solution containing—
Ammonium sulphate ............... 0:
2 gramme
OCMC MI OTIC Mewes swectee acc of aie esac 0:2 os
Potassium hydrogen phosphate... 0-1 y
Magnesium sulphate ............... 00555
Hermous SUP Mates cess sceacsee eet 004 ,
were placed in an Erlenmeyer flask plugged with cotton-wool in the usual
way; to this 1 gramme of calcium oxalate was added, together with, in some
cases, a small quantity of other organic nutrient, and the flask and its
* ‘Zersetzung der organischen Stoffe,’ 1897, p. 217.
30 Mr. A. D. Halland Dr. N. HJ: Miller. [Marae
contents were sterilised; when cool they were seeded with 0:2 gramme of
partly dried surface soil recently drawn from Plot 2 on Broadbalk Field, and
the flasks placed in a dark cupboard at the ordinary laboratory temperature.
The results are summarised in Table XV, and serve to show that the soil
XV.—Bacterial Decomposition of Calcium Oxalate.
l
Refer: | CaCo3 found |
ence | Added to nutrient | Duration, | from |Reaction| State of nitrogen compounds
No solution at starting. days. 1 gramme | at end. at end.
: CaC,0,. |
¥ | |
46 | No soil added ......... 79 — =
50 | No calcium oxalate ... 79 0-015 Neutral
61 5 % baa 176 | trace Es Strong nitrite, slight nitrate.
Ammonia.
44 | Neutral ...............065 "9 0-263 “3 | Nitrate, no nitrite nor am-
monia.
62 ios) ude Sates eeerene 176 OGG i 5 Nitrate, slight nitrite, no
| ammonia.
43 | Slightly alkaline ...... 73 0 428 4 Strong nitrite, nitrate.
64 3 5 oe 176 0-274 | 4, Slight nitrite, strong nitrate,
no ammonia.
42 +0:2 glucose, neutral 79 0-105 ¥ No nitrite nor nitrate.
58 | +02 ,, 5 78 N)05F} |) gg No nitrite nor nitrate. Am-
monia. Glucose gone.
52 | +02 ,, alkaline 73 0-184 | Ss Little nitrite, no nitrate, no
} ?
glucose nor ammonia.
48 +0°2 peptone ......... 80 0°183 59 Nitrite, strong nitrate, no am-
| monia nor organic matter.
SOR Oe es) gi stasiiean 84 0-184 | “ Strong nitrite and nitrate,
some ammonia, no pep-
| tone.
47 | +0:2 calcium humate 82 Og || 5 | Nitrite and nitrate, no am-
| _ monia.
60 | +02 F ; 176 O1G5 | Slight nitrite, strong nitrate
? > § x g 5)
no ammonia.
63 | +02 «4, 5 176 O11440" Pe—,, Slight nitrite, strong nitrate,
no ammonia.
contains one or more organisms which are very effective in converting
calcium oxalate into carbonate. The mechanism of the reaction is being
further studied; in the present connection the experiments are sufficient to
show the existence of other agencies of a bacterial nature engaged in restoring
calcium carbonate to the soil.
The destruction of nitrates by bacterial action, with the evolution of the
nitrogen as gas, the change commonly known as “ denitrification,” is always
attended by the production of a carbonate of the base with which the nitric
acid was combined, but as any calcium carbonate formed in this way would
only replace the calcium carbonate consumed in the previous nitrification
there would be neither gain nor loss to the soil. As also denitrification is
most likely to take place in the lower subsoil where the oxygen of the soil
1905. | On the Retention of Bases by the Soil. 31
gases has been exhausted, any calcium carbonate re-formed in this way would
not appear in the analyses set out, which only extend to the depth. of
27 inches.
It is, however, clear that manuring with organic manures, the growth of
clover and other leguminous plants which leave behind a considerable residue
of roots and stubble particularly rich in calcium oxalate, the débris of plant
tissues which accumulates in the soil of grass land, all go to maintain the
stock of calcium carbonate, which in its turn is being as constantly drawn
upon for nitrification and for the neutralisation of the other acids produced
during the bacterial decay of the carbon compounds the soil receives.
Doubtless in all soils containing only a minimal amount of calcium
carbonate under natural conditions these various actions have reached an
equilibrium, since the increase of any one only tends to bring into play the
factor which limits it (the rate of nitrification, for example, will be slowed
down as the available base in the soil becomes scarce), but also accelerates
the operation of some action in the opposite sense; even the one irrevocable
loss by drainage and removal of crop will probably be balanced by the
calcium salts coming into solution through the continued weathering of the
soil particles. In the main, however, the original stock of calcium carbonate
in the soil circulates continually between plant and soil without suffering
appreciable loss. It is only under particular conditions, such as the use of
ammoniacal manures, or the setting up of anaérobic conditions through lack
of drainage, thus allowing the formation of organic decay acids but not their
final oxidation to carbonates, that the soil will develop an acid reaction and
become infertile.
Summary.
The chief points brought out in the course of the investigation are as
follows :—
(1) Arable soils which contain upwards of 1 per cent. of calcium carbonate
are subject to a normal loss of that constituent in the drainage water
amounting to about 800 lbs. to 1000 Ibs. per acre per annum.
(2) The loss is increased by the use of ammoniacal manures by an amount
equivalent to the combined acid of the manure. The loss is diminished by
the use of sodium nitrate or organic débris like farmyard manure.
(3) The growth of plants normally returns to the soil a large proportion of
the bases in the neutral salts which the soil provides for the nutrition of
plants.
(4) The calcium oxalate and other organic salts of calcium present in
32 Miss J. E. Lane-Claypon. On the Origin, etc., of [June 16,
plant residues are converted by bacterial action in the soil into calcium
carbonate.
(5) The return of base by the growth of plants and the production of
calcium carbonate by the decay of plant residues are sufficient to maintain
soils neutral which are poor in calcium carbonate, and to replace the bases
which have been consumed in nitrification and similar changes.
On the Origin and Life History of the Interstitial Cells of the
Ovary wn the Rabbit.
By JANET H. LANE-CLAYPON.
(From the Physiological Laboratory, University College. Communicated by
Professor E. H. Starling, F.R.S. Received June 16, 1905.)
[PLATE 1.]
The majority of the investigators of the subject consider that the cells of
the germinal epithelium arise by differentiation of the peritoneum, and
become embedded in the subjacent mesoblast, there being probably a dual
process, namely, the downgrowth of the cells themselves and a simultaneous
upgrowth of the subjacent mesoblast.
The fate of the cells thus embedded has given rise to much discussion.
All observers agree in stating that they give rise to the ova, and most
observers consider that they give rise also to the follicle cells; but de
Foulis (8),* Schrén (16), and Wendeler (20), believe these cells to be derived
from the connective tissue.
Pfliiger (15) and Waldeyer (18), although differing in regard to the develop-
ment of the ovary, both consider that the germinal cells give rise to the
cells of the follicular epithelium, there being most probably a previous division
of the original cells.
Nagel (14) also agrees that the follicle cells are derived from the germinal
epithelium. Balfour (4) believed that some of the cells of the egg-clusters
became ova by differentiation, and he described besides a number of small
cells, of which some formed the follicular epithelium and the others probably
either served as foodstuff for the rest, or eventually themselves formed ova or
follicle cells.
Bih' r(7) c seribes the formation of the follicular epithelium by the
* Th se numbers refer to the entries in the bibliography at end. a
1905.| the Interstitial Cells of the Ovary in the Rabbit. 33
streaming inwards from the periphery of some of the cells of the germinal
epithelium.
The changes connected with ovogenesis have been very fully described by
v. Winiwarter (19). They may be briefly summarised as follows. The
germinal cells of the second invagination are rather small and show a nucleus
with some lumps of chromatin, being also rather granular. These he calls
protobroque cells of the a variety. These divide, giving rise to other
protobroque cells a, and also to a b variety. These last divide again
giving rise to more cells of the 0 variety and to a new form of cell, deuto-
broque. The last are larger, and the nucleus more transparent. The deuto-
broque cells give rise to the ova by nuclear differentiation by means of the
following stages. 1. The chromatin breaks up into fine filaments, which are
distributed over the whole nuclear area; this is the /eptotenic stage. 2. The
filaments become gradually massed together until they show as a compact
lump at one side of the nuclear area. This transformation is the synaptenic
stage, which is succeeded by 3, the pachytenic. Here the filaments become
again spread out, but they are much coarser than in the previous stages
The 4th stage, or diplotenic, is so called on account of the tendency of the
chromatin strands to lie in pairs. In the final or dictyate condition the
chromatin is distributed in a reticulum over the greater part of the nuclear area
Balfour describes protoplasmic masses of young ova where the cells appear
fused, and he suggests that one of these ova may grow at the expense of the
rest. Wan Beneden (5) describes multinucleated masses in the ovary of the
adult bat, which he suggests may give rise to an ovum and its follicular
epithelium.
The formation of follicles, which proceeds rapidly, gives rise in the ovary to
two zones, an external or parenchymatous zone in which the follicles lie,
and an interstitial vascular zone; these have been described with some modi-
fications by various workers and for different animals. (His(11), Waldeyer,
Born (6), Macleod (13), Van Beneden.)
The question of the post-natal formation of primordial ova has been the
subject of many isolated observations. Pfliiger believed he had evidence of
the return of the ovary to the tubular formation at the rutting season, the
object of the return being the formation of fresh ova. Waldeyer believed
that all ova were formed in the young animal, and for this reason called all
ova “ primordial ova.”
Schrén noticed an increase in the number of clear cells, presumably ova,
near the periphery in cats and rabbits at the rutting season, and in women at
the menstrual periods. Koster (12) describes prolongations of epithelium
with formation of fresh ova and follicles in the ovaries of evera’ -ecently
VOL. LXXVIL—B. ; D
34 Miss J. E. Lane-Claypon. On the Origin, etc., of [June 16,
pregnant women; Wagener(17) records the thickening of the germinal
epithelium near the attachment of the Fallopian tube in the pregnant bitch, a
condition which he thinks denotes the formation of ova.
Amann (1) describes the presence of follicles in the ovaries of a woman of
65, where there was incipient cystadenoma, the follicles being in all stages of
formation by means of invagination of the germinal epithelium. The interest
of this observation lies in the age of the woman and in the apparent
formation of fresh ova consequent on the stimulus caused by the incipient
eystadenoma.
V. Winiwarter was not able to trace any of the stages of ovogenesis in any
of the adult ovaries he examined, and considers this a necessary condition for
the formation of ova. As far as the literature goes, we may consider the ovary
to be formed by the embedding in the underlying mesoblast of the cells of
the germinal epithelium, the embedding being brought about by a process of
ingrowth of the cells and of upgrowth of the mesoblast. The cells thus
embedded are oogonia, which give rise to ova by division, as also to the
follicle cells, the future ova undergoing considerable nuclear trans-
formation before reaching the condition of the fully-formed primordial ovum.
The post-natal formation of primordial ova has been recorded in certain
cases, but there is not much evidence either in favour of or against it.
Olject of the Investigations.—Certain features which I observed in the
interstitial cells of the ovaries of rabbits at alate period of pregnancy led me
to study the origin of these cells. This question would appear to have been
neglected by previous workers on the ovary. The formation of an internal
ovarian secretion (cf. Andrews (3)), which by analogy with the interstitial
gland of the testis might be presumed to be derived from the interstitial
cells (¢f. Ancel and Bouin (2)), gives considerable interest to their origin.
This was studied by examining (1) the ovaries of rabbits from the twentieth
day embryo up to those of the young rabbit about three weeks after birth ;
(2) the ovaries of pregnant rabbits at all stages.
Methods.—It is not easy to find a really good fixing agent for ovaries,
especially adult ovaries. Hermann’s, Flemming’s (strong formula), Pod-
wyssoski’s and Altmann’s fluids, were all used. The last was found satis-
factory for cytoplasm, but the sections obtained with the other fixatives were
not good. The tissue was osmicated outside and insufficiently fixed inside.
Finally,* Gilson’s fluid was used exclusively for all nuclear figures, and a
* Gilson’s fluid = abs. ale., 1 part ;
glacial acetic, 1 part ;
chloroform, 1 part ;
the whole saturated with sublimate.
1905.] the Interstitial Cells of the Ovary in the Rabbit. 35
mixture of sublimate (saturated) 4 pints, formol 1 pint, and 1 per cent. glacial
acetic for other purposes.
The sections fixed in Gilson’s fluid were stained with iron hematoxylin, or
heemalum ; those fixed in the other solution stain well either with iron hema-
toxylin, hemalum and eosin, or toluidine blue and eosin.
Changes in the Cells of the Germinal Epithelium in the Immature Rabbit.
The origin of the germinal epithelium from the peritoneum by a process of
differentiation has been so fully shown by several observers, that it will not
be necessary to deal any further with the origin of the germinal cells. Also
it has been shown that these cells become embedded in the underlying
mesoblast ; this state of affairs is seen in an embryo of the twentieth day.
The ovary is by this time a definite organ; it is intensely vascular, showing
large blood spaces, especially in the parts lying immediately round the
mesoblastic core. At this period the main mass of the germinal cells is
situated peripherally, only a few isolated ones having penetrated into the
core, which last is sending processes of connective tissue in between groups of
germinal cells. Of these there are present a large number of protobroque and
a few deutobroque ; also a certain number of mitotic figures, but these are not
numerous. (See Plate 1, fig. 1.)
From this time onward until after birth the changes in the ovary, as seen
under the low power of the microscope, are not striking; there are more
deutobroque cells, characterised by their transparent appearance, and there is
an increase in the number of mitotic figures.
Studied under the high power of the microscope some of the deutobroque
cells are seen to have entered upon the early stages of ovogenesis, and to have
reached the leptotenic stage. There are large numbers of round cells showing
a nuclear structure differing from either the protobroque or the deutobroque
cells. The mitoses are chiefly found near the periphery, and the greater
number of them seem to be taking place in the large cells. I do not
altogether agree with v. Winiwarter on tke question of mitosis in these cells.
In the first place there appears to be very little distinction between the
varieties of protobroque cell, a and 0, and I shalJl not dwell upon it. The
mitosis in the protobroque cells does not appear to be sufficient to account for
the large number of deutobroque cells which are formed, and my observations
are to the effect that by far the greater number of mitoses are taking place in
the deutobroque cells themselves. Each class of cell divides, the protobroque
less copiously than the deutobroque, giving rise to two cells of their own
variety. There can be no doubt that the deutobroque cells are modified
D 2
36 Miss J. E. Lane-Claypon. On the Origin, etc., of [June 16,
germinal cells, but I hope to show that the process is one of differentiation,
and not of division.
The protobroque cell is the type of the original germinal cell ; it is small,
generally oblong or oval, and contains a Jarge nucleus. The nucleus shows
a number of chromatin masses of varying sizes, and the whole nuclear area
gives a general impression of granulation (represented by shading in the
figures (Plate 1, fig. 2 (a)). There are no chromatin filaments.
The deutobroque cell is very much larger, and the nucleus has for the most
part a strikingly transparent aspect, the granular appearance noticed in the
protobroque cell being confined to the periphery of the nuclear area. The
chromatin is quite differently arranged; there are one or two irregular
chromatin masses, and strands showing nodular enlargements where they
intersect. See fig. 2 (/). ‘
Sections of a young ovary very soon before or after birth show a large
number of cells whose nuclei exhibit every phase of transition between these
two varieties. These changes in the nucleus may be classed broadly into
three divisions :—
1. The chromatin masses become fewer and larger.
2. There is considerable formation of chromatin strands.
3. The granular appearance gradually passes away from the centre of the
nucleus towards the periphery, leaving the centre clear.
Some of these changes are shown in fig. 2 (0), (c), (d), (e). In the first
stage the whole cell becomes rounder, as also the nucleus, and the chromatin
has begun to aggregate, and there are traces of strands passing away from the
masses. These features increase in intensity until there are only a few
chromatin masses, but the strands are passing between them and intersect in
parts. The granular appearance has: begun to leave the centre, which is clear.
A further process on these lines brings the cell into the typical deutobroque
condition. It would therefore seem that the change from the protobroque
type is accomplished by means of transformations in the nuclear area,
accompanied by a growth in size of the cell, and it is unnecessary to suppose,
under these circumstances, that mitosis is also a method of formation. The
protobroque and deutobroque cells are therefore all oogonia, either potential
or actual, the transition from the one class to the other being probably
accomplished by processes of nuclear differentiation.
At this period in the life of the ovary there is no appearance which could
be characterised either as egg-tubules or egg-clusters; there are large
collections of epithelial cells bounded centrally by the mesoblast, which
presents the appearance of connective tissue. This tissue penetrates but
slightly into the region lying peripherally to the main central core, but careful
1905.] the Interstitial Cells of the Ovary in the Rabbit. 37
inspection shows that there are a few fine processes pushing their way
outwards and more or less enclosing large numbers of germinal cells. The
latter are of all shapes and sizes, from the typical protobroque to the deuto-
broque type.
By the third day after birth the general configuration of the ovary has
changed very considerably. There is still the central mesoblastic core, but
the germinal cells have become more marked off than in the embryo, presenting
the appearance of a definite zone of germinal epithelial cells. The cells are
arranged, especially in the more central parts of the zone, in the form of solid
rods or clusters, several cells thick, which press their rounded ends into the
central mesoblast. Some of them might fairly easily be mistaken for tubules
without a lumen, and there can I think be little doubt that these are what
Pfliiger took for tubules. Around the periphery the tubular formation is not so
marked, the cells lying in irregular aggregations (fig. 3). As the mesoblast is
centrally situated those parts of the germinal zone lying towards the centre
get divided up earlier than the more peripheral parts, which retain the
formation of an earlier stage. This lagging behind, as it were, of the periphery
is quite characteristic of all the changes taking place in the young ovary; it
applies to the formation of tubules and clusters, to the processes of ovogenesis,
as pointed out by v. Winiwarter, as well as to the formation of interstitia
cells, which will be dwelt upon later on.
From this time onwards up to about the twelfth day after birth the changes
in the general contiguration of the ovary are brought about by an amplification
of the processes already described, namely, continued upgrowths of connective
tissue, cutting off the tubules and clusters. The connective tissue likewise
presses into the larger collections of germinal cells, thus cutting them off and
dividing them again into smaller portions, so that as time goes on the clusters
near the central parts consist of less cells, but are present in much greater
number, while those parts more peripherally situated are in a somewhat
earlier stage, the cluster formation being still fairly evident close to the
periphery as late as the sixteenth day.*
Before proceeding to the changes in the egg-clusters about the fifteenth
* Too much stress, however, should not be laid upon the exact date of the young ovary
in relation to its structural aspect. There seems to be an appreciable difference in the
extent to which the ovary is developed in different animals about this age. v. Winiwarter
does not describe any ovary between, the tenth and the eighteenth day, because he does not
consider the changes to be sufficiently striking to call for any description. Of two
litters of rabbits I found slight differences in the ovaries of the same date, the sixteenth
day, the changes being rather more advanced in one than in the other, and both were
almost as advanced as v. Winiwarter’s figure of the eighteenth day. The differences are
probably determined by the varying nutrition of the animal, as also possibly by the kind
of rabbit, some being far more advanced in outward aspect at this age than others.
38 Miss J. E. Lane-Claypon. On the Origin, etc., of [June 16,
day, the transformations which have been taking place in the deutobroque
cells must be briefly touched upon, but they have been so fully described by
v. Winiwarter that a lengthy exposition is quite unnecessary. I shall adopt
his nomenclature throughout. It has already been stated that almost
immediately after birth changes begin to take place in the deutobroque cells,
which enter upon the leptotenic phase, the transformations beginning centrally.
This is succeeded by the synaptenic, and by the third day there are already
a very great number of this variety.
The leptotenic phase is characterised by the absence of visible nucleolus,
and by the spreading out of the chrcmatin in the form of fine filaments over
the whole nuclear area. This stage is evidently only a further step in the
differentiation which has already taken place. It has been shown that the
change from the protobroque to the deutobroque type is accomplished by the
chromatin masses becoming gradually broken up into strands. In the
leptotenic phase the process is merely carried further. Whereas in the
deutobroque stage there are still one or two chromatin masses which have not
become broken up into strands, in the leptotenic this is not the case, the whole
chromatin being present in the form of filaments. The transition stage can
be seen in an ovary of a few days after birth (fig. 6(1)). These filaments
become gradually aggregated, passing to the synaptenic state. The leptotenic
condition is a very fugitive one, whereas the synaptenic, owing presumably to
the great variety of aspects through which the aggregation passes is very
much more prolonged. The synaptenic is succeeded by the pachytenic, where
the filaments are coarser, then by the diplotenic, and this in its turn by the
dictyate condition, which is the typical nuclear appearance of the young ovum.
These changes pass gradually outwards, and by the tenth day even the cells
quite at the periphery have passed through the earliest phases, whilst the
central cells are reaching the final ones. By the fourteenth day there are a
certain number of dictyate nuclei towards the centre. At this stage the
central mesoblastic core is becoming obliterated, the egg-clusters of either
side of the ovary very nearly meeting. The clusters are much smaller,
having been split up by the ingrowing mesoblast. The number of dictyate
nuclei now increases at a surprising rate, there being a great number by the
fifteenth day, and a still greater number by the sixteenth day, by which time
the clusters are almost indistinguishable, except round the periphery, their
place having been taken by dictyate cells, some of which now show a
surrounding follicular epithelium. There are also collections of small more or
less rounded cells lying in between the young follicles, but not forming any
part of the follicular epithelium. These are the interstitial cells of the ovary,
and I propose now to trace their origin in detail.
1905.| the Interstitial Cells of the Ovary in the Rabbit. 39
Origin of Interstitial Cells—By about the tenth day the ovogenetic
processes in the central egg-clusters are at their height, and continue in
this condition, passing through the various phases, until about the fourteenth
day, when, as already stated, some have reached their final stage. Through-
out the whole period, however, there are in almost all the clusters some three
or four cells, or perhaps more, which remain in the ordinary deutobroque
state, and do not undergo any of the ovogenetic phases, the number of these
being greater in the peripheral clusters than in the central ones; by the
fifteenth and sixteenth days, when the number of dictyate nuclei is increasing,
there are few, if any, of the deutobroque cells to be found in the central parts,
but instead, there is an increasing number of the small round cells already
referred to. As the days pass on the number of the former decreases,
and the number of smaller cells increases. Thus, there is throughout the
ovary, but in different parts at slightly different periods, a reduction in the
number of deutobroque cells, which have remained unchanged, and an
increase in the number of small round cells.
Examination of sections of the fourteenth, fifteenth, and sixteenth days
near the centre of the ovary, leaves no room for doubt that some of these
cells form the follicular epithelium, gradually passing towards and arranging
themselves around the young ova. At the fourteenth day the number of
these cells to be found near the centre is not nearly sufficient to form the
follicular epithelium for the large number of young ova, while near the
periphery there are many more than would appear to be necessary for tne
requirements of this part. By the fifteenth day the number of these cells
near the centre has increased very largely, still more so by the sixteenth day,
by which time many of the young ova are surrounded with follicle cells, and
there are also the collections of these cells already referred to. Their number
has meanwhile diminished somewhat at the periphery. The appearance in the
intermediate parts gives the key to the whole question. Here are seen large
numbers of these cells streaming inwards from the periphery and making
their way between the ege-clusters of the periphery towards the centre,
where the cluster formation can now be scarcely recognised. Here they
arrange themselves around the young ova, or pass into little groups by
themselves. These groups are the first beginnings of the real interstitial
tissue of the ovary, and mark the commencement of the adult aspect of the
organ.
There are thus two main points to be emphasised at this period in the life
of the ovary. First, the passage inwards of a large number of cells from the
periphery, and secondly, the commencement of the adult formation by the
formation of young follicles, and the appearance of interstitial cells.
40 Miss J. E. Lane-Claypon. On the Origin, etc., of [June i6,
This passage inwards of cells from the periphery was noticed by Biihler ;
he realised that the number of small cells near the centre during the height of
ovogenesis was not enough to provide a follicular covering for all the young
ova which were there, and he describes the streaming inwards of the cells
from the periphery, and their passage to the young ova, around which they
arranged themselves, and formed the follicular epithelium.
Balfour noticed that in the later periods of ovogenesis there were present
too many of the small cells, like the follicle cells, for them all to become
arranged around the ova and give rise to the follicular epithelium. He was
at a loss to account for the destiny of these supernumerary cells, and supposed
that they must either eventually become ova or follicle cells, or be used up as
food-stuffs for the other cells.
It seems to me, however, that these cells, supernumerary as far as the
follicular epithelium is concerned, are in reality very important. They form
the groups which represent the interstitial tissue of the fully-formed ovary,
and thus, far from being unimportant, are absolutely essential for the
performance of the functions of the ovary.
The question which now arises is, where do these cells come from, and
what is their history of formation ? It has already been indicated that the
number of unchanged deutobroque cells varies inversely with tle number of
these cells, since these last are greater in number in the region where there
are most deutobroque cells present, namely, at the periphery, especially in the
region of the poles, and the high power of the microscope reveals the fact that
these cells are indeed metamorphosed deutobroque cells.
The ordinary deutobroque cell presents one or two irregular chromatin
masses, from which pass out filaments of varying degrees of coarseness and
fineness with nodules at their intersections. The centre of the nucleus
is Clear, whilst around the periphery is the granular appearance already
described. See fig. 2(/). In the ovary of about the eighteenth day the only
regions where these cells are to be seen in any appreciable numbers are
round the periphery and at the poles. They stand out even under the low
power on account of their general transparent aspect as compared with the
surrounding cells, and also in many cases on account of their rather larger size.
There are also cells whose transparency is not so great, but which show up
quite markedly in contrast to the rest and are rather smaller in size than the
more transparent ones. These cells are transformation stages between the
deutobroque and the ordinary interstitial type, and the process resembles very
much in the inverse order that which has been already described for the
deutobroque formation from the protobroque.
The first stage is the gradual massing of the chromatin into irregular masses
1905.|] the Interstitial Cells of the Ovary in the Rablit. Al
and the thinning of the chromatin strands, which become rather less in
number, as do also the nodules (fig. 4(c)). At the same time the granular
appearance extends gradually towards the centre, although it is not until
quite a late stage that it reaches the centre itself (fig. 4 (f) and (g)). The size
of the cell becomes gradually less, and the amount of protoplasm relatively
greater.
The retraction, as it were, of the filaments and strands towards the
chromatin masses is very much more marked in the cells of smaller size,
where there is a tendency for the masses to pass towards the periphery, leaving
the centre clear (fig. 4 (d) and (e)).
These changes continue until nearly all the chromatin is massed, the masses
becoming rounder as the process goes on. There are always traces of strand
formation left, in contrast to the protobroque nucleus, where it is markedly
absent. Thus the small cell derived by differentiation from the deutobroque
cell does not return to the characteristic protobroque type, but shows traces of
its intervening deutobroque condition in the shape of strands of chromatin,
and nodules on the strands. See fig. 4 (g).
The cells, once reduced in size, become true ovarian cells, and may either
function as follicle cells or as interstitial cells (fig. 4 (2) and (£)).
Thus we find the following processes taking place in connection with the
formation of the mature ovary. The cells of the germinal epithelium become
embedded in the underlying mesoblast, and, once there, may either undergo
differentiation, or apparently may remain in the protobroque condition. If
the former be its fate, it must undergo nuclear transformation, together with
growth in size, until it reaches the deutobroque stage. Arrived at this
condition it probably divides, although possibly this is not an essential, and
the two cells formed by this division are of the same type. There are now
two courses open for the cells thus produced ; they may undergo the nuclear
transformations of ovogenesis, and become primordial ova, or they may rest
for a time, and finally undergo regressive transformations, becoming either
follicle cells or interstitial cells. Every cell of the germinal epithelium is
probably a potential ovum, relatively very few remaining in the protobroque
state, although some may still be seen at the periphery in ovaries of the
eighteenth day. Incomparably the greater number pass to the deutobroque
state, preparatory doubtless to the formation of ova. All cannot become ova,
for the other forms of cell are necessary for the maintenance of the ovarian
functions ; possibly, therefore, only the most robust cells, and those which are
most conveniently situated for obtaining nourishment undergo the ovogenetic
changes. This would seem to be borne out by the fact that many more of
the central cells, which are nearer their food supply, undergo ovogenesis, than
42 Miss J. E. Lane-Claypon. On the Origin, etc., of [June 16,
of the peripheral ones. The rest of the cells which are not able, for one
cause or another, to undergo these changes, appear to remain quiescent for a
while, until finally they regress and pass into a condition of subserviency to
the needs of those which have become ova. Both follicle cells and inter-
stitial cells are, however, still potential ova. They have passed through the
initial stages, and only need enlargement and nuclear transformations in order
to become ova, should the appropriate stimulus be given. This chance is not
given to the follicle cells. As soon as the follicle begins to grow, they
multiply rapidly, and probably provide, by their disintegration, the follicular
secretion upon which the ovum feeds and grows. In the ripe follicle of the
rabbit there is almost complete disintegration of the membrana granulosa, and
the remains of the discus proligerus is presumably extruded with the ovum,
perhaps serving it as food material prior to its fertilisation, and subsequent
attachment to the uterine wall. The interstitial celis, however, have possi-
bilities before them, being still capable of carrying out any function belonging
to the true ovarian cell.
All the true ovarian tissue is derived from the germinal epithelium, this
tissue forming in the adult rabbit by far the greatest part of the whole
ovary. There is relatively little mesoblast, which subserves solely the
function of support and of nutriment-carrier to the rest of the organ. We
may, therefore, look upon the whole ovary as consisting of two classes of
cells and of two only, namely, (1) those derived from the germinal epithelium
and performing all the ovarian functions, and (2) those derived from the
original mesoblast, which are supporting and vascular.
There remains only one feature to be dealt with in the immature ovary,
one that has already been described by Balfour, namely, the protoplasmic
masses formed by the aggregations of young ova. In the ovary of the
sixteenth day the ova are all separate, but a day or two later this is not
the case. There are now a large number of these masses of various sizes.
They appear to consist of two, three, four, or even five young ova, to judge
by the number of nuclei seen, but it is impossible to distinguish any trace
of cell-boundary between them. Balfour suggests that these may either
form as many ova as there are nuclei, or that one ovum may develop at the
expense of the rest. This last point of view appears to be the more probable.
It is evident that the massing takes place subsequent to the formation of
the young ova, since it is not seen until after the appearance of the ova,
and it would appear rather purposeless if they merely separated again a
little later on. Moreover, in these masses one or two of the nuclei often
look as if they were disappearing by gradual dissolution, and it is, therefore,
probable that they will all ultimately serve as food-stuff for the one ovum
1905.| the Interstiival Cells of the Ovary in the Rabbit. 43
whose condition happens to have been best, and will, therefore, survive in
the struggle for existence.
This cannibalism on the part of the young ovum is not surprising, if the
life of an ovum be considered. It is really but the normal condition of
the cell at all its stages of development ; it grows and fattens at the expense
of other cells. In the young ovary it is starting its first stage of growth
and must devour other cells; later on, when it grows during the growth
of the follicle, it lives upon the follicle cells, and later still, when, after
fertilisation, the ovum in its extended sense refers to the young fcetus, it
lives on the material provided by the cells of the maternal organism.
This massing of cells and subsequent demolition of some of them for the
benefit of one will be again dealt with in connection with the ovary of the
pregnant rabbit.
Changes in the Ovary during Pregnancy.
The young ovary, after the period when it has reached a stage where the
general aspect is that of an adult ovary, enters upon a period of slow
growth, during which there is a continual formation of a considerable number
of follicles, which having reached a state of partial maturity then begin to
atrophy and finally disappear, leaving only a faint trace of their former
existence in the shape of a scar.
Having reached sexual maturity, the ovary becomes subject to periodic
influences, of the nature of which little, if anything, is known. According
to Fraenckel (9), they are intimately connected with the hypertrophy of the
mucous membrane of the uterus. The sum total of the influences at work
results in the production of “heat,” which occurs in the rabbit about once
a fortnight, but the external changes in the vulva by which this is judged
take place very gradually, so much so, that in the spring and summer time,
when breeding is most prolific, the adult rabbit is scarcely ever out of one
or other stage of “heat.” It is fairly certain, therefore, that whatever
changes may take place in the ovary during “heat,” the condition recurs too
frequently for these to be very marked. This does not refer in any way
to the formation of the corpora lutea of “heat,” which are, of course, very
definite. It has recently been stated by Heape (10) that unless impregna-
tion occurs the ripe follicles of “heat” do not burst, in which case,
presumably, there can be no formation of corpora lutea. If this is the case
it would seem that there can be no such thing as the corpus luteum of
“heat,” and the changes in the ovary during this period must be considered
to consist merely of those taking place before sexual maturity, only rather
more marked, namely, the formation of follicles, but after puberty these
44 Miss J. E. Lane-Claypon. On the Origin, etc., of [June 16,
reach the ripe state, since they will burst if impregnation occur, whereas
this is probably not the case in the immature ovary. There would then, on
this view of the case, be no ovulation except in the impregnated rabbit. It
is quite possible that the additional stimulus of impregnation may hasten
the bursting of the follicle, but it seems somewhat unlikely that without
impregnation there should never be ovulation.
The changes resulting in the production of “heat” are obviously those
preparatory to a possible pregnancy. Fertilisation appears to be in itself
a stimulus, and sets up general hypertrophy of the entire genital apparatus,
producing likewise an improved condition of the animal; as to the mechanism
of the production of this hypertrophy, however, our knowledge may be said
to be nil, and we are reduced to classifying the whole as the changes brought
ubout by the stimulus of pregnancy.
Naked Eye Changes——F¥raenckel describes and figures very accurately the
naked eye changes in the pregnant ovary of about the fifteenth day in his
paper on the function of the corpus luteum.
These changes are very striking, and indicate in themselves some very
definite alteration or increase in the function of the gland; apart from the
formation of the corpora lutea, there is an immense increase in absolute
size, the gradual occurrence of which will now be described.
The ovary of the non-pregnant rabbit is a small yellowish body, lying
on either side against the posterior abdominal wall, a little below the kidney.
It is usually about 4 inch in length and thin, being slightly wedge-shaped
in transverse section and rather pointed longitudinally at either end; upon
its surface may be seen clear round spots, showing the locality of the larger
follicles, some of which, if they are nearly ripe, may even project slightly
from the surface.
The bursting of the follicles and fertilisation lead to the formation of the
corpora lutea, the so-called “true” corpora lutea of pregnancy, and the
growth of these bodies during the early period are undoubtedly the most
characteristic feature in the naked eye appearance of the ovary. If the
pregnancy be one with a large number of foetuses, the ovary often looks
gnarled, so large and numerous are the excrescences produced upon its
surface by these bodies. If these, however, be cut off, and if the organ be
carefully examined at about the fourteenth day, when the corpora lutea are
at their maximum state of development,* it will be readily seen that the
ovary itself has increased in size, quite apart from the formation of the
lutein tissue. The whole gland has a more swollen and rather less compact
aspect; it is larger both in length and girth, and the wedge-shape of the
* Of. Fraenckel, loc. cit.
1905.| the Interstitial Cells of the Ovary in the Rabbit. 45
transverse section is less marked; there are also in many cases fewer
follicles in an advanced condition than in the non-pregnant state. Just
at this period the energies of the gland have apparently been directed rather
to the formation of the lutein tissue than new follicles.
From the fourteenth to the eighteenth day the corpora lutea remain at
their maximum, and then begin to diminish rapidly in size. Instead of
being very vascular whitish bodies, projecting in many cases to the extent
of three-quarters of their whole extent beyond the surface of the gland,
they gradually diminish both in size and vascularity, until by about the
twenty-second day of pregnancy they are merely elevations on the surface,
showing the faintest possible trace of vascularity; this diminution continues
steadily until, a little while before birth, the locality of these striking
features of the fourteenth day of pregnancy is only seen by the presence of
an opaque whitish circular area upon the surface of the ovary. The changes
are so marked that it is possible after a little experience to diagnose very
approximately the previous duration of the pregnancy from the appearance
of the corpora lutea.
Whilst these external changes are taking place in the lutein tissue, the
rest of the ovarian tissue has been also undergoing changes, which, if not
so striking in appearance, are none the less evident. It has already been
stated that the organ at the fourteenth day shows marked increase in size
apart from the corpora lutea; whereas, shortly after this period, these bodies
begin to diminish in size, the reverse takes place in the rest of the ovarian
tissue; and whereas growth of the ovary as a whole has been slow up to the
present, it now becomes rapid and continues until close upon the time of
parturition.
By about the eighteenth or twentieth day all trace of wedge-shape in
cross-section has completely gone, and the organ is nearly circular, the girth
is much greater, and this increase extends right up to the poles. These
changes become more and more marked, until at about the twenty-sixth day
the organ is well over an inch in length, sometimes about 1} inches, showing
a proportionate increase in its other measurements, and having a shape very
much resembling a spindle with blunted ends. The number of clear round
spots has meanwhile been increasing rapidly, so that in the majority of
cases the greater part of the surface is taken up either by them or by the
round whitish patches, which mark the spots where the corpora lutea have
been projecting above the surface. The formation of follicles appears to be
somewhat inhibited during the rapid growth of the corpora lutea, but to
be resumed with greater energy when these have reached their maximum
development. At the time of parturition there are a large number of
46 Miss J. E. Lane-Claypon. On the Origin, etc., of [June 16,
follicles which have almost reached full maturity, and it is a well-known
fact that rabbits can be readily fertilised immediately after parturition.
The gland, although soft, is not in any way brittle, and in spite of its
great general enlargement retains on the whole the same shape, the most
marked change being that from the wedge-shaped to the circular transverse
section.
Changes in Size of the Interstitial Cells——This great increase in size must
be the result either of a large numerical increase, or of a very great increase
in the size of the individual ovarian cells. The latter is at any rate the
main, if not the only factor concerned, the change in size of the cell under
the microscope being so marked as to attract attention even apart from any
actual measurement.
The measurements were made with a micrometer eye-piece, gauged against
a micrometer slide, this method being found quite sufficiently accurate for
the purpose. It was not intended to record exactly the size of each
individual cell, but rather by taking the measurements as accurately as
possible of a large number, to find the average increase in size at different
stages of pregnancy. In taking the measurements considerable selection was
exercised in the cells measured; only those whose area in section was
approximately circular, and where the nucleus was centrally situated being
used, as it was hoped by these precautions to obtain measurements passing as
nearly as possible through the centre of the cell. The measurements are
given below of 10 cells from each date of pregnancy, but this does not by
any means represent the number actually measured, but the same figures
recur again and again, and the average works out to almost precisely that
given.
Towards the end of pregnancy there is considerable difficulty in finding
the right kind of cells to measure, nearly all of them being angular and
irregular in outline, giving as a whole somewhat the appearance of a
tesselated surface. The changes in general aspect of the sections, produced
by the change in the size of the cells, will be returned to later on.
1905.| the Interstitaal Cells of the Ovary in the Rabbit. . 47
Interstitial Cells of a normal Rabbit.
Diameter at 14th day of pregnancy.
Diameter in mm.
00162
0:0180
00171
0:0189
00180
00153
00180
00198
00171
0:0189
01773
Average = 0:°0177 mm.
Diameter at 18th day.
mm.
0:0252
0:0279
0:0288
0:0279
0:0270
0:0288
0:0270
00252
0:0270
0:0279
0:2727
Diameter at 22nd day.
mm.
0:0324
00315
00324
00315
00315
00306
00360
00351
00342
00315
0:3267
Average ="0°0272 mm.
Average’ = 0:0326 mm.
= 32°6'u
mm.
0:0225
00216
00245
0°0207
00252
00216
0:0225
0:0270
0:0252
0:0243
0°2349
Average = 0:0234 mm.
23°4 w
Diameter at 20th day.
mm.
00542
0:0333
0:0306
0:0506
0:0297
00315
0:0324
0°0315
0°0324
0:0333
03195
mm.
0:0306
0:0288
0:0297
0:0342
0:0270
0:0333
0:0360
0:0351
0:0315
0:0324
03186
Average = 0:0319 mm.
= 21D jn
Diameter at about 26th day.
Average = 00318 mm.
= 318 p
48 Miss J. HE. Lane-Claypon. On the Origin, etc., of [June 16,
Diameter just before
birth, at 28th or Diameter a few hours
29th day. after parturition.
mm. mm.
0:0288 00270
0:0315 0:0279
0:0270 0:0279
0:0297 00270
00288 Average = 0:0298 mm. 00270 Average = 0:027 mm.
0:0315 = 2058 0:0261 = 2
0:0288 0:0252
0:0306 00279
00315 0:0270
00306 0:0279
0:2988 i 0:2709
Diameter after Diameter after
3 days’ lactation. 6 weeks’ lactation.
mm. mm.
0:0270 0:0180
00252 00171
0:0279 00162
00270 00180
00270 Average = 0:0268 mm. 0:0171 Average = 00171 mm.
0:0252 == ASM 00153 gg ILO 0,
0:0279 00180
00261 00171
0:0270 , 00162
0:0279 00180
0:2682 01710
Tabulating these results, one gets—
Approximate age of pregnancy. Diameter of cells in p.
OX(= normal) Weer eee e eee EO)
TA tho daly. cceuAvanssespossene sasenneeee 23°4
Sth. se eaceeccee cere capensis: 27-2
QO th....,, wien eerdeaoneucemee seep ese Bley
VAIS GRRE ESTO 3 Sexe, Seki oop OSB AGUS OP 32°6
Bit, 5. palevoge ace ee eee reer ae 31°8
ShortlivsbetonesoinhineeeeetPeereeees 29°8
‘3 aTGCI A ye nian ween nae 27:0
3 Cans PDE SG aRARN ENE 26°8
6 weeks ,, s lc ill esl Rae al eal
1905.| the Interstitial Cells of the Ovary in the Rabbit. 49
Taking the radius of the cells it is seen that the increase in its length in the
cell during pregnancy is from 8°5 to 16°3 or very nearly double.
If the volume of the sphere be taken as 4 7 . r? and the cell be taken as a
sphere, the ratio of the non-pregnant cell to the cell at the maximum size
attained during pregnancy becomes almost exactly 1:7, which would allow
sufficient enlargement of the ovary to account fully for the increase in size.
It is not to be supposed that all the cells enlarge to the same extent, but it
may reasonably be supposed that they enlarge to about five times their
normal size. This will account for the enlargement of the whole ovary, and
there would seem therefore to be no necessity to seek any further cause of
the enlargement of the ovary during pregnancy.
The only other possible cause which suggests itself at once is of course the
division of cells, but although I have examined some hundreds of sections of
pregnant ovaries, I have not found any trace of this happening. In giving
the above figures I do not wish to suggest that the measurements are
absolute. They are subject most probably to individual variations, depending
possibly upon the number of fcetuses in each pregnancy, and on various
other circumstances. The ovaries in question were, however, taken quite
haphazard in regard to all external causes, which allows some scope for
differences in the ovary, and the results are fairly definite. They show a
great increase in the size of the ovarian interstitial cells during pregnancy,
and that the main increase is reached by about the twenty-second day, and is
sustained until just before birth, when there is a slight diminution in size.
In this connection there is one feature to be dealt with, namely, the shape
of the cell. Up to about the twenty-fourth or twenty-fifth day it is not
difficult to find approximately spherical cells to measure. After this period,
however, the difficulty of doing so becomes very great, if not impossible.
The cells are angular and seem crushed together, and I would suggest that
possibly the cells may be really still undergoing slight increase in size, but
that the capsule having almost reached its maximum stretching capacity does
not admit of the desired expansion, and the cells instead of being spherical
become more closely packed in order to find room for the additional bulk,
filling in as it were the interstices rather than causing au increase in size in
the spherical direction.
The rounded appearance is resumed very shortly after birth, and there is
also a slight decrease in size. Why there should be a decrease before birth
is a point upon which I feel it is impossible to offer any suggestion. The
mechanism of the production of labour is a question upon which very little
is definitely known; if, however, it be the function of the ovary to cause the
adhesion of the foetus to the uterine wall (Fraenckel), a function carried out
VOL. LXXVII.—B. iE
50 Miss J. E. Lane-Claypon. On the Origin, etc., of [June 16,
presumably by means of the interstitial cells, since these probably furnish
the internal secretion of the organ, it seems not impossible that the
diminution in size may be indirectly connected with the onset of labour.
The Formation of “ Primordial” Ova from the Interstitial Cells.
In addition to the increase in size there are other changes taking place in
some of the interstitial cells near the peripheral parts of the ovary, during
the later third (approx.) of the period of gestation. It is a matter of
common histological knowledge that over the surface of the ovary there is
a layer of epithelial cells, roughly about two cells deep, although varying
slightly in thickness at different places. Immediately below this is a layer
of tissue in which are embedded the primordial ova in their early stages,
when they have not yet acquired a follicular epithelium or when that
epithelium is not very highly developed. There are in addition groups of
small ovarian cells which will eventually, as occasion arises, form the follicle
cells for the primordial ova. This whole layer together with the germinal
epithelium varies very considerably in thickness in different animals, the
variation having possibly some relation to the age of the animal under
investigation (cf. v. Beneden).
In the non-pregnant animal and in the early periods of pregnancy, there is
a fairly sharp boundary between these outer layers and the deeper lying
interstitial cells. By about the twentieth day of pregnancy this state of
affairs is seen to be gradually changing, and some of the interstitial cells are
becoming surrounded by the connective tissue of the inner layer and thus
getting cut off from their fellows below. Whether this is brought about by
the passing outwards of the cells themselves or by the growth inwards of the
connective tissue is very difficult to decide quite satisfactorily ; but I think
it is reasonable to suppose that both processes are involved. It has already
been shown that there are two means whereby the germinal cells of the
embryo become embedded in the subjacent mesoblast, namely, by an
ingrowth of the germinal cells and by a simultaneous upgrowth of the
mesoblast lying below. Here we have an analogous condition, but the
positions are reversed; the germinal cells are now inside and the mesoblast
outside.
This process, which is beginning to be evident about the twentieth day,
continues throughout the rest of pregnancy, so that as the days go on more
cells become cut off and press outwards, in many cases reaching almost to
the periphery. The number of cells thus cut off varies appreciably in
different animals, probably depending upon the age of the animal, but it is
not excessive at any time; I have never found more than three or four rows
1905.] the Interstitial Cells of the Ovary in the Rabbit. 51
of cells cut off, and these rows do not form continuous layers round the
ovary (vide fig. 5).
About the twenty-third or twenty-fourth day, and in ovaries of later dates
of pregnancy, a somewhat striking feature about some of these cells is that
they are no longer mononucleated; two nuclei are frequent, three quite
common, whilst in some cases there may be as many as six. These nuclei
are not massed together as in a giant cell, but lie separate in the cell
protoplasm. The latter is very much greater in amount than in an ordinary
interstitial cell, and is irregular in outline. The appearance of these
multinucleated cells suggests that they have been derived from the fusion of
the same number of interstitial cells as there are nuclei in the cell. It will
be remembered that van Beneden pointed out this appearance in the bat’s
ovary, when he found in some cases as many as eleven nuclei, and he
suggested that possibly one of them grew at the expense of the others, whom
it used as food, or that one might become an ovum and the others the follicle cells.
Examination of a large number of these cell masses shows that in many
cases there is undoubted atrophy of one or more of the nuclei going on. In
some there is a clear space where a nucleus might have been expected, in
others the nucleus stains very faintly or only in parts, whilst there is usually
one nucleus which stains intensely, especially in the iron hematoxylin
specimens, and in which the staining, even after extreme differentiation, is
still so dark as to remove all possibility of tracing any nuclear structure.
This points to some difference of metabolic condition, and the conclusion
seems obvious that this nucleus is growing strong at the expense of the
others ; one is reminded of the protoplasmic masses described by Balfour in
the young ovary and to which reference has already been made in this paper.
Here we have a number of potential ova (for the fact has already been
emphasized that all interstitial cells being derived from the germinal
epithelium are potential ova) massed together, of which the nucleus of one
of them grows at the expense of the others, which it uses as food material ;
in the young ovary the end-product is a primordial ovum. In the pregnant
ovary the end-product is likewise a “primordial” ovum. The cells of these
aggregations are all quite clearly ordinary interstitial cells, and the surviving
cell is also an interstitial cell differing only in the intensity of its staining
reaction,
It has I hope been conclusively shown, in the earlier part of this paper,
that the interstitial cells have all been derived from the cells of the germinal
epithelium, and have all passed through the deutobroque condition, and it
has been pointed out by v. Winiwarter that if there is to be ovogenesis
subsequent to the first great ovogenetic period, the cells which are to become
E 2
52 Miss J. EH. Lane-Claypon. On the Origin, etc., of [June 16,
ova must pass through the requisite nuclear changes. Also it is obvious,
although this is not a point which he brings out, that there is a very great
difference in size between the primordial ovum and the interstitial cell in a
non-pregnant animal, and it is therefore necessary for the cell to enlarge at
some period of the transformation. This requirement is fulfilled, as has
already been shown, in the case of the pregnant ovary. The interstitial cell
of the non-pregnant ovary has approximately a diameter of 17 y, but
increases up to 29 w or even rather over 30 ~ in the pregnant animal. The
size of a primordial ovum before it begins to grow, preparatory to becoming a
Graafian follicle, is very constant; I have taken measurements of a large
number of ova both in the young ovary and in the pregnant as well as the
non-pregnant animal, and the average diameter is 27 yw, the diameter reached
by the interstitial cells about the eighteenth day of pregnancy.
It is about the twentieth day of pregnancy that the cutting off of the
interstitial cells towards the periphery begins to be noticeable—that is to
say, shortly after they have reached a diameter about equal to that of a
primordial ovum. It is not, however, until a little later that the cells thus
cut off begin to show any nuclear differentiation, in fact this is perhaps best
seen in the ovary of a rabbit whose time of parturition has almost arrived.
These changes are identical with those taking place in the deutobroque cells
of the young ovary during the period of ovogenesis. The only difference lies
in the fact that whereas in the pregnant ovary the process is taking place
only at the periphery, and in relatively very small numbers, in the young
ovary there may be 20 or 30 nuclei undergoing changes in the same field.
The fact of their presence at all in the pregnant ovary is, however, all proof
that is necessary for the formation of ova. It is not for a moment to be
supposed that any formation of fresh primordial ova after the first great
period should take place to anything like the same extent. Probably the
actual changes only occur over a period of a few days, commencing about the
twenty-fifth day of pregnancy, or rather earlier, and extending probably to a
little after parturition. In the young ovary the changes do not commence
until after birth, and some of the cells have completed their changes by about
the tenth or eleventh day, the process being probably considerably less
lengthy than this for the individual cells, and taking still less time, if any-
thing, in the pregnant rabbit, where there is obviously a state of stimulation
during the whole period of pregnancy.
The first change passed through by the nucleus of an interstitial cell, which
has passed to the periphery in order to become an ovum, is shown in fig. 6 (1).
The nucleus shows chromatin filaments, in the middle of which are seen
irregular lumps of chromatin. (In the diagrams the analogous stage of the
1905.| the Interstitial Cells of the Ovary in the Rabbit. 53
young ovary has been given side by side with that in the adult, and does not
call for any special description.) Thisis a transition form from the interstitial
nucleus to the leptotenic stage in the process of ovogenesis, and appears to be
brought about by the breaking up of the nuclear chromatin into an immense
number of filaments. The arrangement of the chromatin in the interstitial
cells is, as a rule, discrete either in a rather loose reticulum or round the
edges, usually the former.
The first change is therefore the formation of fine filaments. The leptotenic
stage of v. Winiwarter is brought about by the enlargement of the nuclear
area and the spreading out of the filaments over this increased space, thus
producing a looser arrangement which consists of fine filaments with a rather
nodular appearance where they intersect (fig. 6(2)). This state would appear
to be a very fugitive one (as observed likewise by v. Winiwarter), judging by
the rarity of its occurrence. It is quickly passed through, and the nucleus
enters upon the synaptenic condition (fig. 6 (3)). This stage occupies much
longer than the last, and a relatively large number of nuclei are found in this
condition, which has many modifications. The filaments at the leptotenic
stage are spread out over the nuclear area, whilst at the final synaptenic the
chromatin is massed into a lump at the side of the nucleus. All stages may
be traced both in the adult pregnant ovary and the young ovary, but only
the most characteristic phase is figured, namely, that where a very appre-
ciable amount of massing has already proceeded, the mass being connected to
the sides of the nucleus by a few very fine filaments.
The massing completed, there seems to be a rearrangement of the
chromatin, and it becomes spread out again, but this time the filaments are
thicker. This is the pachytenic stage (fig.6(4)). The number of nuclei found
in this stage is less than in the synaptenic, but still there are a fair number
in various conditions. The filaments are so markedly thicker and more bulky
generally that it is impossible to confuse it in any way with the leptotenic
phase. The chromatin does not fill the nucleus quite so much as in the young
ovary, but I have found sections where this was more the case than in the
one figured; moreover in some the chromatin seems to have a more continuous
disposition than is here represented.
The transition stage between the pachytenic and dictyate or final stage is
not, according to my observations, quite analogous to v. Winiwarter’s, and I
rather hesitate to call it diplotenic, as the duality of the filaments is not well
marked (sce fig. 6 (5)); the chromatin is still arranged in thick strands, and
there is some trace of nucleoli, whilst at the same time there are a very few
thinner nodulated strands, foreshadowing the condition called by v. Winiwarter
dictyate, and which represents that of the young ovum.
54 Miss J. E. Lane-Claypon. On the Origin, etc., of [June 16,
The nucleolus in the dictyate condition (fig. 6 (6)) is very definite, and the
chromatin is arranged more or less all over the nuclear area (which is now
very large), and shows a number of small nodules both at what appear to be
free ends and at the points of intersection. There can, in fact, be not much
doubt that the changes taking place are identical with those seen in the
young ovary, which lead to ovogenesis, and therefore it would appear that
ovogenesis also takes place in the adult animal during pregnancy.
Previous observers on this subject appear to have all considered that
formation of ova must be accomplished by means of fresh invaginations of
germinal epithelium, and those who thought they saw invaginations con-
cluded at once that there was therefore a formation of ova in later life, whilst
those who failed to find them denied the possibility on this account. My
observations show that fresh invaginations of the germinal epithelium are
not a necessity, but that the “invagination” has taken place already in the
embryo. The invaginated cells of the germinal epithelium give rise to all
the cells of the true ovarian tissue, which are all capable of functioning in
any true ovarian capacity—that is, they may become ova or follicle cells, or
interstitial cells, and most probably also lutein cells, their destiny appearing
to be a matter of chance. The interstitial cells, however, are still capable of
becoming ova, and of undergoing the changes requisite for ovogenesis should
the appropriate stimulus be given. This stimulus is supplied when the animal
becomes pregnant, and the ovarian cells enlarge in size. Towards the end
of the time of pregnancy some of them press towards the periphery and
undergo the necessary changes, becoming true ova. Thus every pregnancy
would seem to be a stimulus for the next, in the way of providing new ova,
although even of the relatively small number found probably very few ever
reach maturity.
Conclusions—Summing up the conclusions reached in this paper we find—
1. That a large number of germinal cells become embedded in the subjacent
mesoblast. Of these the great majority undergo transformations up to a
certain stage. This stage having been reached, they may pass through the
necessary processes of ovogenesis, or they may become modified to form either
follicle cells or interstitial cells, this last process being the chief fate of the cells
near the periphery, whilst ovogenesis is that of the more centrally situated
ones.
2. The interstitial cells are thus potential ova, capable of becoming ova
should the appropriate stimulus be given.
3. This stimulus is provided by pregnancy, during which period the
interstitial cells undergo enlargement in size, exceeding that of a primordial
ovum.
1905.] the Interstitial Cells of the Ovary in the Rabbit. 55
4, About the twenty-third day some of the interstitial cells become cut off
near the periphery and pass through the nuclear transformations of ovogenesis,
becoming true ova.
I wish to express my deep obligation to Professor Starling, under whose
supervision this research has been carried out, and without whose never-
failing assistance, interest, and sympathy at each step of the work
it would have been impossible to carry out the investigations described
above.
Also I desire to thank Mr. H. G. Plimmer for his kindness in giving me
much valuable information in regard to the carrying out of the histological
details.
BIBLIOGRAPHY.
Amann, ‘ Verhandlung der Ges. Deutsch, Naturf. und Arzte in Miinchen,’ 71 Vers.,
vol. 2, 1899.
2. Ancel and Bouin, ‘Comptes Rendus Soe. de Biol.,’ 1903 and 1904.
3. Andrews, ‘ Journ. of Obst. and Gyn. of British Empire,’ May, 1904.
4. Balfour, ‘Quart. Journ. Micros. Science,’ 1878.
5
6
=
Van Beneden, ‘ Arch. cle Biol.,’ vol. 1, 1880.
. Born, ‘ Arch. f. Anat.,’ 1874.
7. Biihler, ‘Zeit. f. Wissen. Zool.,’ 1894.
8. De Foulis, ‘Quart. Journ. Micros. Science,’ 16, ser. 3, 1876.
9. P. Fraenckel, ‘ Arch. f. Gynik.,’ vol. 68, part 2, 1903.
10. Heape, ‘ Roy. Soc. Proc.,’ April, 1905.
11. His, ‘ Arch. f. Mikros. Anat.,’ vol. 1, 1865.
12. Koster, ‘ Meded. der Kon. Akad. der Wetenschappen,’ 2 reek, teel 3, 1868
13. Macleod, ‘ Arch. de Biol.,’ vol. 1, 1880.
14. Nagel, ‘ Arch. f. Mikros. Anat.,’ vol. 31, 1888.
15. Pfliiger, (1) ‘ Ailgem. Medizin. Centralzeitung,’ 1861 and 1862 ; (2) Uber die Eierstécke
der Saugethiere und des Menschen,’ 1867.
16. Schrén, ‘Zeit. f. Wissen. Zool.,’ vol. 12, 1863; ‘Inaug. Dissert. der Med. Facultat
zu Erlangen,’ 1862.
17. Wagener, ‘Arch. f. Anat. u. Phys. Anat.,’ Abthg., 1879.
18. Waldeyer, ‘ Hierstock und Ei,’ Leipzig, 1870.
19. v. Winiwarter, ‘ Arch. de Biol.,’ Liége, 1900.
20. Wendeler, ‘ Martin’s ‘Die Krankheiten der Eierstécke und Hier,’ Leipzig, 1899.
For further literature on the early history of the ova see :—
Von Baer, ‘De Ovis Animalium et Hominis Generi,’ 1827.
Barry, ‘ Phil. Trans.,’ London, 1838.
Billroth, ‘ Miiller’s Arch.,’ 1856.
Bischoff, ‘ Bau des Menschlichen K6rpers,’ Sémmering, 1842.
Borsenkow, ‘ Wiirz, Naturwissen. Zeitschrift,’ vol. 4, 1863.
Coste, ‘ Embryogénie Comparée,’ p. 80.
De Graaf, ‘De Mulierum Organis Generationi Inservientibus,’ 1668.
Grohe, ‘ Virchow’s Arch.,’ vol. 26.
56 Miss J. E. Lane-Claypon. On the Origin, etc., of [June 16,
Henle, ‘Handbuch der System. Anat.,’ vol. 2, 1866.
Kolliker, ‘ Entwickelungs-geschichte des Menschen und der Hoheren Thiere.’
Langhans, ‘ Virchow’s Arch.,’ vol. 38.
Letzereich, Pfliiger’s ‘Untersuchungen aus dem Physiol. Lab., Bonn,’ 1865.
Purkinje, ‘ Encyclopidisches Wérterbuch,’ vol. 10, Artikel “ Hi,” Berlin, 1834.
Romiti, ‘Arch. f. Mikros. Anat.,’ vol. 10, 1874.
Rouget, ‘Comptes Rendus de I’Acad. des Sciences,’ 1879.
Spiegelberg, (1) ‘ Nachricht d. k. Ges. der Wissenschaft,’ Géttingen, 1860 ; (2) ‘ Virchow’s
Arch., vol. 30, 1864.
Stricker, ‘Wiener Akad. Sitzungsbericht. Math. Naturw. Klasse,’ 2 Abthg., vol. 54,
1866.
Valentin, ‘ Miiller’s Arch.,’ 1838.
R. Wagner, ‘ Beitrige zur Geschichte der Zeugung und Entwickelung, p. 42.
Wharton Jones, ‘London and Edinburgh Phil. Mag.,’ No. 39, Sept. 1835, p. 209.
DESCRIPTION OF PLATE 1.
Fie. 1.—Ovary of 20th day embryo (rabbit). Fixed in Gilson’s fluid :—
@ = protobroque cells.
b= ; (in mitosis).
ce = deutobroque cell.
d = connective tissue cell.
e = position of mesoblastic core.
Fie. 2.—Cells in ovaries of rabbits just before and after birth. Fixed in Gilson’s
fluid :—
a = protobroque cell.
b |
|
‘ > = transition forms between (a) and (/).
e
J = deutobroque cell.
Fic. 3.—Ovary of three days old rabbit showing formation of egg-clusters.
sublimate solution :—
a = protobroque cell.
6 = deutobroque cell:
C= - in mitosis.
d = leptotenic stage in ovogenesis.
Fie, 4.—Cells in ovaries of young rabbits. Fixed in Gilson’s fluid :—
@ = deutobroque cell in ovary of three days old rabbit.
b= % 3 about 18 days old rabbit.
¢ |
, = transition stages from deutobroque to ovarian cell in ovary
t | 18 days old rabbit.
9g 3
A = interstitial cell
= Policia cell } trom ovary of young rabbit (18 days).
Fixed in
of about
Lane-Claypon. Roy. Soc. Proc., B. vol. 77, Plate 1.
1905.| the Interstitial Cells of the Ovary in the Rabbit. 57
Fie. 5.—Ovary of rabbit about 22nd day of pregnancy. Taken from the cortical
region. Fixed in sublimate solution :—
a = germinal epithelium.
6 = primordial ovum.
e = multi-nucleated interstitial cell.
d = interstitial cell, becoming isolated.
f = connective tissue.
g = modified germinal cells.
Fie. 6.—1—6 are taken from the ovaries of rabbits in the later stages of pregnancy.
la—6a from ovaries of young rabbits : showing ovogenetic changes for comparison with
1—6. Fixed in Gilson’s fluid :—
1 = transition from interstitial to leptotenic phase in pregnant ovary.
la = ” 9 young 9
2 = Leptotenic phase in pregnant ovary.
2a = ” yoans ”
3 = Synaptenic phase in pregnant ovary.
3a = i young %
4 = Pachytenic phase in pregnant ovary.
4a = ” young " £h)
5 = Diplotenic phase in pregnant ovary.
5a = 9 young os
6 = Dictyate phase in pregnant ovary.
6a = ” young ”
58
Ferivlity in Scottish Sheep.
By Francis H. A. MarsnHatt, M.A. (Cantab.), D.Sc. (Edin.), Carnegie
Fellow, University of Edinburgh.
(Communicated by Professor EH. A. Schafer, F.R.S. Received August 10, 1905.)
My attention was first directed to the subject of fertility by Mr. Walter
Heape, to whom I am much indebted.
Experiments have been described in agricultural publications on the effects
of different methods of feeding and general treatment upon wool or meat
production; but excepting, so far as I am aware, for Mr. Heape’s report on
“ Abortion, Barrenness, and Fertility” in sheep in the South of England for
the year 1896 to 1897,* no systematic attempt has been made to deal with
the factors which influence fertility either in the sheep or in other animals.
Numerous experiments, however, are annually conducted by flock-masters
for a practical object, and it has been thought desirable to put the results of
some of these on record with a view to making comparisons, and in the
hope eventually of reaching definite conclusions upon this subject.
That differences in food and environment exercise an influence over
fertility in the sheep as in other animals has long ago been recognised,+
and recently attention has been called to the wide range of variability
in the sheep’s sexual capacity, this animal showing a complete gradation
between the moncestrous condition and the most extreme degree of
polycestrum.}
Asa preliminary step in an investigation on fertility in sheep, it was decided
to issue a schedule of queries addressed to various flock-masters chiefly in
the East of Scotland. The present communication consists of a condensed
account of some of the information contained in their replies.§
The preparation and issue of the schedule was undertaken by the Highland
and Agricultural Society of Scotland, under whose auspices the work is being
carried on. I am under no light obligation to the members of this society
for their co-operation, as well as to all those gentlemen who have supplied
* “ Abortion, Barrenness, and Fertility in Sheep,” ‘Journ. Roy. Agric. Soc.,’ vol. 10,
1899. ‘Note on the Fertility of Different Breeds of Sheep,” ‘ Roy. Soc. Proc.,’ vol. 64,
1899.
+ Darwin, “ Animals and Plants,” Popular Edition, London, 1905.
Marshall, ‘The Cistrous Cycle and the Formation of the Corpus Luteum in the
Sheep,” ‘Phil. Trans.,’ B, vol. 196, 1903.
§ It is hoped that a full report may be issued next year in the ‘Transactions’ of the
Highland Society.
Fertility in Scottish Sheep. 59
me with information regarding their personal experiences. The com-
paratively small number of schedules issued and returned (the latter being
about 50), rendered it possible to obtain fuller information than would other-
wise have been the case, while the information obtained in this way was in
some cases supplemented by personal conversation or further correspondence.
For the purpose of showing the percentages of lambs per ewes, of barrenness,
and of abortion (Tables I, II, and IIL) among flocks treated in different ways,
these are divided into six groups as follows :—*
Division A.—This includes hill sheep (Scotch Black-faced and Cheviots),
which were kept all the year round on the sides of hills, and received no
sort of special treatment.
Dwision B.—This includes hill sheep (Scotch Black-faced and Cheviots),
which were placed upon better grass at tupping time (7.¢., during the sexual
season) or shortly before.
Division C._—In this division are included half-bred (Border Leicester x
Cheviot) ewes which underwent a process of flushing by being fed on turnips,
cabbages, oats, dried grains, maize, or other artificial food during tupping and
for about three weeks before. The ewes were in most cases merely fed on grass
during the greater part of the year, but received a certain amount of extra
food (turnips, ete.) during the latter part of pregnancy (7, usually from
about the beginning of the year).
Division D.—This includes two flocks of Cheviot ewes which were flushed
at tupping time but were fed on grass during the rest of the year.
Table I—Number of Lambs per 100 Ewes.
locks: Under | 90 | 100 | 110 | 120 | 180 | 140 | 150 | 160 | 170 | 180 | 190 Total.
90 p.c.| p. c. | p.c. | p. ¢. | p. ¢.| p. c.| p. c. | p. c.| p. c. | p.c.| p.c.| p. c
Division A 2 10 2 1 — | — | — | — | — | — | — |] KH 15
noe BL = Li hop Sf} dp ee tS ee pee Sf 4
5 GC] = —{;—|;—j]1 i | = | 2 3 i | =} 2 10
ye. oD: — — | — | — 1 1 — | — | — | — | | 2
» = — | — | — | = | =| 2 1 Bi} al 1 7
»p of a ee SS 1 2 2)/—|]— 6
Total...... 2 10 3 1 3 2 3 4 8 3 1 3 44,
iT {
The numbers represent the numbers of flocks, the total being 44. The percentages are the
percentages of lambs per ewes in the different flocks. The flocks are arranged in six divisions,
according to the methods of feeding, as explained in the text.
* The variation in the number of flocks in the three tables is due to the flock-masters
not having supplied complete information im all cases. Some flocks, therefore, are
included in one table but not in another.
60 Dr. F. H. A. Marshall. [ Aug. 10,
Table I1.—Percentage of Ewes that Aborted.
Flocks. | None. ae lp.c. | 2p.c | 3p.c. | 4p.c. | Sp.c. | Total.
Division A ......... — — 6 3 2 2 iL 14
Bias ESS ed vane a 3 == = = TL a 4
is nal OP Aas 4 3 2 1 = ce 10
Bee aD ecitccne — | 1 = _— — — 2
Sy iets! Ohnescmeneee 2 3 1 1 = = 7
op We yrecsiatasene 2 1 1 = — 1 _— 5
Total .-....- 8 11 11 5 | 2 4 1 42° |
The numbers represent the numbers of flocks, the total being 42. The percentages are the
percentages of ewes which aborted in the different flocks. The flocks are arranged in six
divisions, according to the methods of feeding, as explained in the text.
Table IIJ.—Percentage of Barren Ewes.
Flocks. None. oa lp.c.| 2p.c.|3p.c | 4p.c | 5p.c | 6p.c. | 7p.c. | Total.
Division A| — ay —_ 2 3 — 5 2 iL 14
eBid = us 1 1 1 we il ae 4
we 3 — — 3 1 1 — — — 8
Sad ae 1 1 _ a0 = es as 2
eh ge 2 4 — a —-};-}] =— 7
eS es = 2 1 ee 1 1 = = 5
Total...... 3 1 5 12 5 4 6 3 1 40
The numbers represent the numbers of flocks, the total being 40. The percentages are the
percentages of barren ewes in the different flocks. The flocks are arranged in six divisions,
according to the methods of feeding, as explained in the text.
Division H.—This includes flocks of Border Leicester and half-bred (Border
Leicester x Cheviot) ewes which were placed on better pasture durig
tupping and for some time (usually about three weeks) before, but which
otherwise received no sort of special treatment; in some instances, however,
the ewes received a limited number of turnips during pregnancy.
Diwision #.—TVhis division includes Border Leicester, and half-bred (and a
few Cheviot) ewes which were fed all the year round on grass, receiving no
special treatment of any kind.
Table I shows very clearly that the percentage of lambs was, as a rule,
larger among flocks which underwent a process of artificial stimulation during
the sexual season, while Table III shows that the percentage of barren ewes
was generally relatively less in such flocks. The Cheviot and Black-faced
sheep in Division B which produced less than 100 lambs per 100 ewes
(Table I) are stated to have been unusually unprolific owing to their never
1905. ] Fertility in Scottish Sheep. 61
having properly recovered from the extreme cold in March and April, 1904.
This case, therefore, may be regarded as exceptional. The percentage of
barren ewes in this flock was six (Table ITI).
In the three cases in which the percentage of lambs was over 190
the exact numbers were 191-5 per cent., 193°75 per cent., and 196 per cent.
In the first of these the ewes (which were half-bred Cheviot x Border
Leicester) were fed on grass only, during the previous summer. For three
weeks (during tupping) they were given a full supply of turnips on grass,
and between tupping and lambing (five months) they were given a mixture
of dried grains and turnips, and “lamb food” for three weeks before lambing.
The rams (which were pure Border Leicesters) were given bruised oats during
tupping. No record was kept of the ages of the ewes. One ewe had four
lambs and 12-5 per cent. had triplets.
In the second case the ewes (half-bred) were fed upon Bombay cake,
bruised barley and a little linseed as well as turnips and cabbages during
tupping (after grass), and some turnips were given during pregnancy. The
rams (Border Leicester and Oxford Down) were similarly treated. The ewes
were all three-shear. Flushing with turnips was found to bring the ewes in
season very rapidly. Triplets were produced by 13:5 per cent. of the ewes.
The third case is recorded under Division E, but ought possibly to have
been included under Division C. At tupping time the ewes (which were
half-bred) were put upon better pasture, and between tupping and lambing
they were given some turnips and as much cut hay as they would eat.
Previously to tupping they were fed on grass alone.* The ewes were all ages
up to four-shear. The rams (which belonged to the Border Leicester, Oxford
Down and Cheviot breeds) were supplied with no artificial food at tupping.
The twins appear almost invariably to have been born early during lambing
time, thus showing that the reproductive activity of the ewes is generally
greatest early in the tupping season. Only two returns record that twins
were mostly born late, while 28 state that early twins were the rule, both
among the artificially fed flocks and those which received no special
treatment.
There is abundant evidence also that flushing hastens forward the tupping
time. It has recently been shown that “heat” in animals is almost certainly
brought about by an internal secretion elaborated in the ovaries. It would
appear, therefore, that the artificial feeding exercises a stimulating influence
* Cheviot ewes, kept on the same farm, and treated similarly, produced only 10:0 per
cent. lambs.
+ Marshall and Jolly, “ Contributions to the Physiology of Mammalian Reproduction.
Part Il.—The Ovary as an Organ of Internal Secretion,” ‘ Phil. Trans.,’ B, vol. 198, 1905.
62 Dr. F. H. A. Marshall. Fertelity in Scottish Sheep.
over the secretory activity of the ovaries, while at the same time causing the
Graafian follicles to mature more rapidly and a larger number to discharge
during the earlier cestrous periods in the sexual season.
Regarding the effects of artificial feeding during one tupping season upon
the fertility of the sheep in after years, it has so far been difficult to obtain
precise information. The opinion usually expressed is that flushing is not
detrimental to subsequent fertility unless it is overdone; but in a very few
of the returns the view is stated that the after-effect is adverse. It is also
said that if ewes are flushed one year the process must be repeated the next:
otherwise the ewes tend tv be less fertile than if they had never been flushed
at all.
On the other hand, several of the returns show that sheep which produce
twins one year very frequently bear twins also in the year following. This
seems to occur irrespectively of whether it was the practice to flush the ewes.
It would appear, therefore, that an increased degree of fertility is characteristic
of certain particular ewes.
That fertility is a character which can be inherited admits of no doubt. It
is to be noted, however, that with the breeds considered in this paper, twius
are seldom if ever selected for purposes of tupping, since they generally are
not so well developed, owing to their having had less nourishment when they
were young lambs. It would seem, therefore, that the fertility of these
breeds is diminished owing to the fact that the rams which are probably
naturally the most fertile are the ones which are the least frequently employed
for breeding
63
On the Nature of the Galvanotropic Irritability of Roots.
By AurreD J. Ewart, D.Sc., Ph.D., F.L.S., and Jessie §. Bayuiss, B.Sc.
(Communicated by Francis Darwin, For. Sec. R.S. Received September 7,—
Read November 23, 1905.)
After the contradictory statements of Elfving* that roots curve towards the
positive electrode (anodotropic), and of Muller-Hettlingen,t that they were
kathodotropic, Brunchhorst{ apparently reconciled these contradictory observa-
tions by finding that strong currents, like those used by Elfving, produced a
curvature to the positive electrode, weak ones a curvature to the negative
electrode. The former curvature Brunchhorst considered to be traumatropic
in character, on the ground that it was shown by decapitated roots, whereas
the negative curvature was not. The proof that the galvanotropic irritability
resides solely in the root tip, is, however, quite insufficient, and hence
Brunchhorst’s conclusion does not appear to be justified by the facts. The
methods of the first two investigators leave much to be desired, and although
Brunchhorst’s experiments were, in part, carried out on a klinostat, they are
by no-means perfect. Thus the roots were immersed in water in a closed
vessel, through which the current was passed by means of carbon electrodes.
Apart from the effects due to the gases occluded by the electrodes, and to the
deficiency of oxygen in the water, there would always be a tendency for the
current to run obliquely or longitudinally through the roots, whose tissues
form better conducting media than the surrounding water. This tendency
will be especially pronounced when the roots are not exactly at right angles
to the current, as is practically always the case, and when, as in Brunchhorst’s
experiments, numerous roots are examined at the same time. Finally,
although Brunchhorst gives some data as to the total amount of current
flowing in the circuit, these data afford no evidence as to the actual amount
of current passing through the individual roots. Evidently, therefore, the
supposed positive and negative parallelo-galvanotropism of roots is by no
means satisfactorily established, and accordingly Miss Bayliss undertook to
reinvestigate this subject, under more well-defined and controllable conditions,
and with the results given in brief below.§
The strength of constant current required to produce a curvature is
incredibly small, for using a voltage of approximately 1:3 volts, a resistance
* ‘Bot. Zeit.,’ 1882, p. 257.
+ Miiller-Hettlingen, ‘ Pfliiger’s Archiv,’ vol. 31, 1883, p. 193.
{ Brunchhorst, ‘Ber. d. D. Bot. Ges.,’ 1884, vol. 2, p. 204.
§ Full details will be given by Miss Bayliss in a later paper.
64 Dr. A. J. Ewart and Miss J. S. Bayliss. On the [Sept. 7,
of 100,000 to 150,000 ohms was required in the circuit, so that the current
passing through the 1 to 3 sq. mm. of cross-section lay between 0°0000135
and 0:000009 of an ampere. Even then it was difficult to produce a eurva-
ture without serious injury, or even fatal effects in the case of sensitive roots.
When the platinum electrodes were on opposite sides of the apex, the curva-
ture was always towards the positive electrode. If, however, one electrode
was placed on the non-irritable base of the root and the other to one side
of the apex, the curvature always took place towards the current side, inde-
pendently of which electrode was on the apex. These results were obtained
upon a klinostat into which the current was led by mercury contacts, and
transmitted by platinum electrodes to the stimulated region of the root.
The seedling and wires within the rotating glass cylinder were insulated on a
slab of paraffin wax.
The facts observed suggested that the curvatures were not the result of
any parallelo-galvanotropic irritability, but were due to the accumulation of
the products of electrolysis at the points of application of the electrodes.
Confirmation was obtained by exposing the roots to strong currents (voltage
1 to 4) for short periods (five to eight minutes), and then rotating on a
klinostat, when exactly similar results to the above were given. Furthermore,
if the anodal region was cut out of an electrolysed root and applied to one
side of the apex of another, a curvature was shown to this side. In addition,
the application of minute squares of absorbent paper, moistened with
decinormal acid or alkali, caused curvatures towards the stimulated side,
whereas ordinary neutral paper produced no effect in air saturated with
moisture. When the acid and alkali were applied simultaneously on opposite
sides, the curvature always took place towards the acid side. This corresponds
to the curvature towards the positive (acid) electrode produced by moderately
strong currents. The weakest currents used produced similar positive curva-
tures, and hence Brunchhorst’s negative curvatures cannot be explained
by Weber’s law, as being due to the normal acidity of the root tissues
preventing the stronger stimulating action of the acid coming fully into play
until it accumulates beyond a certain limit.
The curvatures are usually completed in from 6 to 24 hours after exposure
to .the current, but they may be distinctly perceptible within four to six
hours, and may begin in one to two hours, under optimal conditions. Hence
it is not surprising that if the roots are fixed in a plaster cast after stimula-
tion, and rotated on a klinostat for one or two days, a rapid sharp curvature
is produced on freeing the root from the cast, whereas after two to four days
the effect of the stimulation has passed away. All of these curvatures can be
produced without any of the cells of the root being killed, and even when an
1905.|] Nature of the Galvanotropic Irritability of Roots. 65
injury is produced, the curvature is usually towards the injured side, instead
of away from it, as in a true traumatropic curvature.
The curvatures produced by continuous currents appear usually to be
accompanied or preceded by a temporary more or less pronounced retardation
of the average rate of growth inlength. Indeed the latter may be temporarily
arrested for some time after strong stimulation, even when the electric current
produces little or no injury. In such cases negative results may be obtained
as regards curvature.
Finally, using non-polarizable electrodes moistened with cell-sap diluted
with distilled water, no curvatures were produced, whereas similar stimulation,
using platinum electrodes applied to the surface of the root, and with the
non-polarizable electrodes still in the circuit so that the resistance was the
same, gave the usual curvatures according to how and where the electrodes
were applied. With stronger currents and more prolonged exposure, curva-
tures are induced, even when “ non-polarizable” electrodes are used, since the
products of electrolysis may diffuse to the surface of the root, and it is
impossible to prevent the internal polarization which takes place wherever the
current traverses dissimilar saline solutions separated by semi-impermeable
membranes. There is, however, less tendency to injury than with platinum
electrodes.
The irritable and responsive zone extends 4 to 5 mm. behind the apex of
the rootof Vicia Faba and Phaseolus vulgaris. When one platinum electrode
was applied to the non-irritable base of a root, and the other laid flat on the
extreme tip, no curvature was produced in whichever direction the current
was passed. This is presumably due to the products of electrolysis diffusing
evenly and stimulating the irritable regions and cells equally on all sides, for
when the same current was applied transversely behind the apex, a positive
curvature was shown. If the roots were either truly positively or truly
negatively parallelogalvanotropic, they should curve in the above experiment
so as to place the tip parallel to the current, and either against or with its
direction, whenever this does not at first coincide with their tropic
irritability.
The “ galvanotropism ” of roots is therefore due to chemotropic stimulation
by the products of electrolysis, of which the acid is more effective than the
alkali, the latter also being neutralised more or less by the respiratory carbon
dioxide. It is indeed possible that the curvature of the roots of Lupinus
albus in gelatine towards phosphates and carbonates observed by Lilienfeldt*
may be of similar origin, since acid phosphate and alkaline carbonates were
used. That the “galvanotropic” or galvanogenic curvatures are not trauma-
* Lilienfeldt, ‘Ber. d. D. Bot. Ges.,’ 1905, vol. 23, p. 91.
VOL, LXXVII.—B. F
66 Mr. F. Keeble and Dr. F. W. Gamble. Jsolation of [Oct. 6,
tropic in origin is shown by the fact that they may be produced without any
cells being killed. In Brunchhorst’s experiments the electrolysis presumably
occurred in the superficial cells of the roots submerged in water, the tissues
being sufficiently impermeable superficially to the liberated acid and alkaline
ions to allow them to accumulate beyond the minimum for stimulation.
Although the curvature is usually sharp and strongly localised to the point
of application of the electrode, the discriminatory power of the root, as well
as the relation of the rates of growth on concave and convex sides to the
normal rate of growth, suffice to show that the response is a stimulatory one,
and is not due to the direct action of the products of electrolysis, retarding
growth on one side or accelerating it on the other.
On the Isolation of the Infecting Organism (“ Zoochlorella”) of
Convoluta roscoffensis.
By FREDERICK KEEBLE, M.A., University College, Reading, and F. W. GAMBLE,
D.Se., University of Manchester.
(Communicated by Sydney J. Hickson, F.R.S. Received October 6, 1905.)
The present paper gives a preliminary account, (1) of experiments proving
that the green cells (“‘zoochlorelle”) of Convoluta roscoffensis result from
infection from without: (2) of the means whereby the infecting organism
may be cultivated outside the body of the animal: and (3) of the nature of
the infecting organism.
1. Evidence for Infection—In our former papers* we reached the con-
clusion that though direct proof of infection was lacking, the evidence
pointed most strongly to infection as the source of the green cells of
Convoluta. We showed, moreover, that the difficulty in the way of obtaining
direct proof of the origin of these green cells is due to the fact that the mucil-
aginous capsules that invest the clutches of eggs laid by Convoiuta are rarely,
if ever, sterile. Even when adults are washed repeatedly in sterilised sea-
water and caused to lay in sterilised surroundings, their egg-capsules become
covered in time with a varied flora of colourless and of green organisms.
It is therefore necessary to isolate the young at the moment of hatching.
During the present summer we have done this in larger numbers than before
and maintained them in carefully filtered sea-water. Such young Convoluta
* “The Bionomics of Convoluta roscoffensis,’” ‘Roy. Soc. Proc.,’ vol. 72, p. 93, and
‘Quart. Journ. Micro. Sci.,’ vol. 47, p. 363, 1903.
1905.| the Infecting Organism of Convoluta roscoffensis. 67
remain colourless and may be kept in this condition for at least a month
without showing any sign of infection, whilst at any time batches of them
may be caused to become green in one to three days by the addition of sea-
water or of cultures of the infecting organism.
2. The Cultivation of the Infecting Organism.—All attempts to cultivate
green cells taken from the body of Convoluta have failed. Haberlandt made
an unsuccessful attempt, we ourselves were equally unsuccessful, and so also
was Miss Harriette Chick, who brought to the task great experience of such
researches and the most recent methods.
The problem had therefore to be attacked from the other end. If the
green prisoners of Convoluta never escape alive, the only chance of obtaining
the infecting organism lies in catching it before its entrance into the animal.
A scrutiny of many attempts to obtain colourless Convoluta in large quantities
revealed the fact that generally egg-capsules, isolated in sterile water, give
rise to Convoluta which remain colourless for a fairly well-marked period
of two or three weeks. After this time, however, green specimens make
their appearance. Sometimes the number of green animals thus appearing
is few; more often it increases with great rapidity. Such results suggested
that the infecting organism occurs sporadically on or in the capsules; that
it divides freely in this situation; and that after a period of vegetative
division it is liberated in sufficiently large numbers to infect the hundreds
of Convoluta experimented upon.
During the past summer this hypothesis has been put to the test and
found to be correct. Large numbers of egg-capsules were kept in filtered
water and the young Convoluta upon hatching were removed, so that the
vessels contained only empty capsules or capsules the eggs of which had
failed to hatch. These vessels were kept under observation. At the end of
three weeks several minute spherical bodies of a spinach-green colour were
detected. Upon microscopical examination these proved to be colonies of
green cells enclosed by and filling an egg-capsule. During examination
the membrane around such a colony bursts, and the contents, previously
quiescent, swarm out of the capsule, revealing themselves as so many
unicellular flagellated green organisms.
It remained to apply the infection test. Samples of colourless Convoluta
reared in sterilised surroundings were put into the vessel containing these
flagellated cells. They became infected, and in the course of two or three
days exhibited in their tissues green cells identical in character with those
of normal Convoluta roscoffensis.
Similarly, sterilised sea-water containing cultures of these green organisms
is as potent as ordinary unsterilised sea-water in producing infection.
F 2
68 On the Infecting Organism of Convoluta roscoftensis.
Ordinary sea-water or such cultures alike induce infection within a few
days, when added to just hatched Convolutas reared previously under sterile
conditions; whereas the addition of sterile sea-water to samples from the
same stock of Convoluta produces no infection.
3. Nature of the Infecting Organism—Whilst reserving for a detailed and
illustrated account the full description of the organism infecting Convoluta
roscofjensis, we may here briefly refer to its more salient characters and
systematic position. In its adult and holophytic stage, the motile green
organism is ovoid and flattened in front. At the anterior end it possesses
two pairs of similar flagella. A basin-shaped chloroplast envelops the greater
part of the body, and is turned in anteriorly, forming a clear border to a
colourless funnel-shaped area which runs axially for a third of the cell’s
length. A plate-like red “eye-spot” or stigma, with a darker rim, lies
somewhat excentrically a little in front of the middle of the cell ana not in
relation to the flagella. The resting nucleus is spherical. Near the posterior
end of the chloroplast is a large octagonal pyrenoid, provided with a starch
sheath, giving it a somewhat irregular outline. True starch, giving a marked
blue colour with iodine, is present in quantity. A cell-wall is either absent
or of extreme tenuity in the organism when first liberated, but later on a
marked wall of mucilaginous character is demonstrable and may reach a
considerable thickness.
These characters indicate that the green cells of Convoluta roscoffensis are
true alge, belonging to the Chlorophycee and allied to Chlamydomonas. The
presence of four equal flagella suggests that they belong to the genus
Carteria.*
* Blackman and Tansley, ‘New Phytologist,’ vol. 1, p. 23, 1902.
69
Further Observations on the Germination of the Seeds of the
Castor Oil Plant (Ricinus communis).
By J. REYNOLDS GREEN, Sc.D., F.R.S., Professor of Botany to the Pharma-
ceutical Society of Great Britain, and Henry Jackson, M.A., Fellow
and Tutor of Downing College, Cambridge.*
(Received March 22,—Read May 18, 1905.)
About 15 years ago one of the authors carried out a series of researches
on the germination of the seeds of the castor oil plant (Ricinus communis),t
and endeavoured to ascertain the course of the decomposition and utilisation
of the reserve materials which are present in the seed. As the results
of this investigation formed the starting point of the present series of
researches, it will be well at the outset to restate the conclusions which
were then arrived at.
The larger part of the reserve materials of the seeds of Ricinus, which
are laid up in the cells of the endosperm, consists of the well-known castor
oil. The amount varies in different seeds, but it ranges from as little as
50 per cent. to upwards of 80 per cent. There is a considerable amount
of proteid matter in the cells, most of which is found in the so-called
aleurone grains. These have a somewhat intricate structure; an ovoid
mass of phytoglobulin, soluble in 10 per cent. solution of common salt,
surrounds a proteid crystal, soluble in saturated solution of the same salt.
In the grain by the side of the crystal there is a rounded aggregation of
mineral matter, the so-called globoid, long considered to be a double
phosphate of calcium and magnesium, but probably a more complex body
containing its phosphorus in some form of organic combination. According
to Vinest the proteids of the grain are an albumose and a globulin;
in the opinion of Osborne and Harris§ this is not the case, only a globulin
being present, probably identical with the edestin of the hemp seed. There
are other substances present in small amount, but in very trifling pro-
portions when compared with the oil and the proteids.
* The present series of experiments was commenced by me in collaboration with
Mr. W. T. N. Spivey, of Trinity College, Cambridge. After his lamented death in 1901,
Mr. Jackson took his place.—J. R. G.
+ Green, ‘Roy. Soc. Proc.,’ vol. 48 (1890),. p. 370.
t Vines, “ Proteid Substances in Seeds,” ‘Journ. of Physiol., vol. 3 (1880), p. 91.
§ Osborne and Harris, “ Nitrogen in Protein Bodies,” ‘ Amer. Chem. Journ., vol. 25
(1903), p. 335.
70 Prof. J. Reynolds Green and Mr. H. Jackson. [Mar. 22,
The conclusions arrived at in 1888 with reference to the changes set up
during germination were the following :—
“1. The reserve materials in the endosperm of Fucinus communis consist
chiefly of oil and proteid matters, the latter being a mixture of globulin
and albumose.
“2. The changes during germination are partly due to enzyme action, there
being three enzymes present in the germinating seed: one is a protease
resembling trypsin, the second splits the oil into fatty acid and glycerine,
the third is a rennet enzyme.
“3. At least two of these, and therefore presumably all of them, are in
a zymogen condition in the resting seed, and become active in consequence
of the metabolic activity set up in the cells by the conditions leading to
germination, especially moisture and warmth.
“4, The changes caused by the enzymes are followed by others, due to
the metabolism of the cells, these being processes of oxidation.
“5, The embryo exercises some influence on the latter, setting up as it
develops a stimulus probably of a physiological description.
“6. The result of these various processes is to bring about the following
decompositions :—
“The proteids are by the enzyme converted into peptone, and later into
asparagin.
“The oil is split by the glyceride enzyme into fatty acid and glycerine ;
the latter gives rise to sugar, and the former to a vegetable acid
which is soluble in water and in ether, is crystalline, and has the
power of dialysis.
“7, Absorption in all cases takes place by dialysis.
“8. The appearance of starch and of oil in the embryo or the young
plant is due to a secondary formation, and not to a translocation of either.”
FORMATION OF LECITHIN.
The advances in our knowledge of the metabolic processes of plants that
have been made during the interval that has elapsed since the publication
of this paper, and the new methods of experiment that have been introduced,
suggested that the work which was admittedly incomplete and tentative
should be taken up again. There remained especially the question of the
meaning of the reserve supplies of phosphorus and the part which they
take in the general metabolism accompanying germination. The aggregates
of phosphates referred to as the globoids of the aleurone grain undergo a
change during the process, by virtue of which they slowly pass into solution.
1905.| On the Germination of Seeds of the Castor Oil Plant. 71
As this change supervenes upon the development of an acid reaction in
the seeds, it seems not unlikely that it may be caused by the action of the
organic acid which is formed in the cells of the endosperm almost as soon
as germination begins.
On resuming the work a more careful examination of the oily contents
of the endosperm cells led to the discovery that they contained, mixed with
the oil, a certain quantity of a substance, the decomposition products of
which pointed to its being a lecithin (a peculiar fatty body containing
phosphorus). The resting seeds were pounded in a mortar till they formed
a homogeneous paste. This was extracted for some hours with ether in
a Soxhlet’s apparatus, and was afterwards twice extracted further on a
water-bath with absolute alcohol, the flask being fitted with a reflux con-
denser. The alcoholic and ethereal extracts were mixed and evaporated
to dryness at a gentle heat on a water-bath, and the fatty residue fused with
dry carbonate and nitrate of potassium until all trace of free carbon had
disappeared. After cooling, the fused residue was dissolved in water, and
the addition of ammonium molybdate and nitric acid produced a yellow
precipitate, indicating the presence of phosphorus. The quantity of the
latter was ascertained by converting it into magnesium pyrophosphate and
weighing.
A little of the oil was then hydrolysed by boiling with baryta, when
there separated out a flocculent precipitate of a barium salt, which, after
washing and drying, was found to contain no phosphorus. This barium salt
had the characteristic soapy appearance of the stearates. It was found
possible to identify cholin in the endosperm of the germinating seeds, as will
be more fully shown a little later (p. 74).
The only way of estimating the amount of the lecithin present in the
alcohol-ether extracts of the endosperm was to determine accurately the
phosphorus as magnesium pyrophosphate. Great precautions were taken
to ensure the repeated use of very dry ether so as to exclude the possibility
of extracting any inorganic phosphates. A little of the oily residue from
the extraction was incinerated in a platinum dish, and it was found to leave
no inorganic ash. We therefore assumed that all the phosphorus extracted
as deseribed was originally present in the complex lecithin form. Taking
the formula usually given for lecithin (CssHaNPO,), we calculated the
amount of the latter that would be present. Its average amount was equal
to 0°236 per cent. of the dry weight of the seeds.
Several series of experiments were made to investigate the changes in the
fatty constituents of the endosperm during germination. The action of
the fat-splitting enzyme known now as lipase was confirmed, and the early
72 Prof. J. Reynolds Green and Mr. H. Jackson. [Mar. 22,
stages of germination were found to be as set out in the former paper.
Consequently, examinations of the contents of the seeds were made at
certain stages of the germination, and before the process had begun. The
stages were the following :—(1) The seed at the time of the cracking of the
testa, usually after 24 to 48 hours in the soil; (2) the seed with the radicle
protruding for a length of 1 to 2 cm., usually about three days after sowing;
and finally, (3) seeds whose lateral root system had become fairly well
developed. The times at which these stages were reached varied with the
samples of seeds used, and the temperatures at which the germination
took place.
The results of a typical experiment are stated in the subjoined table :-—
Table A.
Oil in seeds. Fatty acid in seeds.
Dry Lecithin
Degree of weight | per cent.
development. of seeds Percentage | Percentage} of weight
used. aa ral of weight Gi ee of weight | of seeds.
WEE of seeds. | “eSB | of seeds.
grammes. | grammes. grammes.
Resting seeds ............ 4°48 3°7115 828 O1 2-2 0°236 |
Seeds just cracking} 4°47 3 016 67-5 0-204 4°6 at |
testa .
Radicle protruding 1—2| 4:17 2-19 52°53 05 ibt) 0-478
cm.
Lateral roots spreading. 3°34 0-789 23 ‘6 0-565 16 ‘89 0 873
Root system estab-
lished
It will be seen that the amount of lecithin diminished during the early
stages of germination, the reserve supply becoming almost exhausted. After
the young seedling had begun to develop, however, there was a gradual
increase in the amount. This increase was maintained during the later
stages and was fairly constant till the endosperm was used up. There was
clearly a consumption of the oil throughout.
The amount of lecithin, though small, varied somewhat in different
experiments. In one series it was in much larger proportion than in that
quoted. The residue soluble in alcohol and ether amounted to 0-9 per cent.
of the weight of the, resting seeds, and in the later stages of germination the
amount present rose to approximately 2 per cent. This quantity, however,
in our experiments was exceptional.
These experiments suggest that in the utilisation of the fatty reserves
lecithin certainly plays a part and, possibly, a predominant part.
1905.| On the Germination of Seeds of the Castor Oil Plant. 73
Lecithin has been shown by Overton * to be a normal constituent of living
cells, and to exercise considerable influence on the transport of various
materials across the limiting layers of the protoplasm. It has no doubt also
a certain, though at present undetermined, nutritive value.
The composition of lecithin is indicated by the change which it undergoes
on hydrolysis, when it is decomposed into stearic (or palmitic or oleic) acid,
glycero-phosphoric acid and cholin.
CuHoNPO, + 3H20 = 2CisH3602 + CsHoPOg + CsHisNO2.
Lecithin. Stearic acid. Glycero- Cholin.
phosphoric
acid.
From this, its constitution has been represented as
J oC 0GiHss
C3H;—-OCOC7Hs; :
\O — PO(HO)0(CH2)2N(CH;),0H
Only a trace of it exists in the resting seed; as it increases during
germination and the quantity remains fairly constant during the whole
period of absorption of the fatty reserves by the seedling, we have evidence
of a formation of it during the germinative processes. The endosperm
contains such substances as may yield the several groups necessary for its
formation. The decomposition of the oil by the enzyme lipase can furnish
the fatty component, belonging to the oleic group, and at the same time the
glycerine of the glycero-phosphoric acid. The phosphorus of the latter is at
hand in the shape of the phosphatic globoids whose solution has already been
alluded to. The ‘nitrogenous body cholin may be looked for among the
products of the decomposition of the proteids of the aleurone grains.
Examination of the contents of the endosperms during germination
ultimately established the presence of all these constituents. The fatty
acids and the glycerine were identified in 1888, and the methods of detection
and estimation were quoted in the former paper. A careful examination of
the phosphates of the globoids, taken for purposes of comparison from seeds
at the respective stages of germination quoted in Table A (p. 72) showed that
their solution proceeded side by side with that of the oil.
No change in them could be observed under the microscope till the testa
was cracking, and the time of its inception varied a good deal. In the early
stages, prior to such cracking, no reaction for phosphorus could be obtained
from a watery extract. The quantity of phosphorus present in the resting
seed was 0:205 per cent. of the dry weight; this diminished in Stages 2, 3,
and 4 of Table A to 0:16, 0:14, and 0-11 per cent. The globoids are decom-
* Overton, ‘ Pringsheim’s Jahrb.,’ vol. 39 (1900).
74 Prof. J. Reynolds Green and Mr. H. Jackson. [Mar. 22,
posed gradually but fairly rapidly during the germination, and in the later
stages contribute to the acidity of the cell-sap, which contains phosphoric
acid.
Search was made in a mass of endosperms for cholin. The germinating
seeds were ground up in a mortar and allowed to stand for some days under
alcohol which was nearly absolute. This was decanted and evaporated to
dryness, the residue being again extracted with absolute alcohol and
subsequently by a mixture of alcohol and ether. These extracts were
mixed and evaporated to dryness, leaving a final residue, soluble in water.
When a strong aqueous solution of this was boiled, decomposition took place,
and a gas was evolved which possessed the well-known ammoniacal and
fishy odour characteristic of tri-methylamine. The decomposition can be
represented by the following equation :—
CH.(OH)CH.N(CH;);0H = CH.(OH)CH,OH + N(CH;).
When to some of an aqueous solution of the residue from the alcoholic and
ethereal extracts a little platinic chloride was added, after standing for some
time the characteristic yellow octahedral crystals of the compound which
cholin forms with platinic chloride separated out. These were soluble in
15 per cent. alcohol, and on combustion yielded a residue of metallic
platinum. We have thus all the constituents of lecithin present in the
germinating seeds.
It was difficult to apply the ordinary tests for lecithin when so large
a quantity of oil was present. Towards the close of the germination,
however, conditions were more favourable, the lecithin being present in
relatively large proportion.
The existence of a proteolytic enzyme of a tryptic nature in the
germinating seeds was shown in the former paper. Among the products of
its action a considerable quantity of crystalline amino-bodies were detected,
though not sufficient for a complete analysis. They separated out from the
concentrated alcoholic extracts, after removal of the sugars, in quantities
that enabled their amino-nature to be proved. The power of the enzyme to
produce these in vitro has already been noted.* We have found the cholin
also to be due to the action of this enzyme. 150 cc. of an extract of the
endosperms of a quantity of germinating seeds was prepared by steeping
them for several hours in water containing 0°2 per cent. of formaldehyde as
an antiseptic. It was then strained through muslin and filtered till it
appeared as a clear straw-coloured liquid. This was divided into two, and
half of it boiled ;to destroy the protease. A quantity of globulin was
* Green, loc. cit., p. 377.
1905.] On the Gernunation of Seeds of the Castor Oil Plant. 75
prepared from a further quantity of the same germinating seeds by
extracting them with a 10 per cent. solution of common salt, and precipi-
tating the proteid by addition of alcohol. The precipitate was rapidly
collected on a filter, washed and suspended in a little water.
The 75 cc. of the extract that had not been boiled was put into a beaker
and 5 ¢.c. of the suspended globulin added; a similar preparation was made
of the 75 c.c. that had been boiled. Both were kept in an incubator at 40° C.
for a week. At the end of that time digestion was complete in the unboiled
preparation, the globulin having disappeared, leaving a morbid solution.
Both were perfectly free from bacteria, the formaldehyde acting extremely
efficiently as an antiseptic. The two digestions were then filtered and the
filtrates evaporated to dryness. The residues were extracted successively
with absolute alcohol and with a mixture of absolute alcohol and ether, each
extraction .being continued for two days. The first alcoholic extract was
evaporated to dryness and the residue again extracted with ether. The two
ethereal extracts were subsequently mixed and evaporated to dryness and
the residue taken up with a little water. There was considerably more of
this residue in the digestion carried out by the unboiled extract of the seeds
than in that associated with the other. To each a little platinic chloride was
added in watch-glasses, and they were set aside. After 24 hours, in both
cases minute crystals had settled to the bottom of the liquid, which were
soluble in alcohol of 15 per cent. concentration. From this solution the
characteristic yellow octahedra slowly settled out, and these gave the same
reactions as those prepared from the extracts of the endosperms. The amount
obtained from the digestion by the unboiled extract was much greater than
that from the boiled one, though the latter yielded some, attributable no
doubt to a certain quantity present in the 75 c.c. of the original extract of
the seeds employed.
The experiment shows, therefore, that the cholin of the lecithin can be
prepared from the proteids of the seeds by an enzyme which is developed
during germination, and is presumably the enzyme already described as a
trypsin.
The similarity of this enzyme to the trypsin of the pancreas is borne out
by the occurrence of tryptophane among the products of its activity both in
the plant and in vitro in the laboratory.
The contribution of material for the synthesis of lecithin does not seem,
however, to be the only result of the decomposition of the fat. There is not
sufficient phosphorus in the resting seeds to enter into the composition of as
much lecithin as the fat would produce. It is, of course, possible that the
lecithin may be decomposed during consumption and part of its phosphorus
76 Prof. J. Reynolds Green and Mr. H. Jackson. [Mar. 22,
set free to combine again, but even then the quantities do not seem to be
proportional. Another fate must attend a considerable quantity of the fat.
To this point we shall return later.
These results suggest that the utilisation of the oily reserves is a much
more complicated process than was supposed. The enquiry took from this
point a wider range, and soon involved the abandonment of the idea that the
separate reserves undergo independent changes during germination.
THE SUGARS OF RICINUS.
A more complete study of the sugar was next undertaken. Du Sablon
showed, in 1895* that it is a mixture of at least two sugars, one of which
has not the power of reducing Fehling’s solution. In our experiments, a
large number of seeds having been germinated, the endosperms were separated
from the embryos and ground to a paste in a mortar. The mass was then
extracted with large quantities of water, by keeping it for some hours in
a steriliser at 100° C., removing the water at intervals till the extract
showed that all the sugar had been dissolved. The extracts were mixed
and concentrated to about one-tenth of their volume. Addition of normal
acetate of lead separated from this extract the acids present, together with
the bulk of the proteids and certain other constituents. These were filtered
off, and the sugars were precipitated from the filtrate by adding basic lead
acetate and ammonia. The precipitate was separated by filtration and
suspended in water, and the lead removed by a stream of sulphuretted
hydrogen. The solution so obtained was concentrated, and the process
repeated, the final solution being then concentrated to a thick syrup, which
showed the presence of two constituents possessing different solubilities in
alcohol. By a repetition of concentration and extraction, the syrup was
ultimately separated into two parts, one of which reduced Fehling’s solution,
while the other did not. Unfortunately the separation did not involve the
complete isolation of the two sugars, as the reducing power of the first fraction
was always increased after boiling with dilute mineral acid. The increase was
not constant in different preparations, a fact which pointed to incomplete
separation rather than to the reducing sugar being of the maltose type.
The second fraction of the syrup was, however, free from the reducing
sugar. Treated with invertase or with a dilute mineral acid it speedily
reduced Fehling’s fluid. A quantity of it was concentrated nearly to dryness
and with some difficulty dissolved in alcohol. Addition of ether to a little
of the solution caused precipitation of the sugar. To the great bulk of the
* Du Sablon, “ Sur la Germination des Graines Oléagineuses,” ‘Rev. Gén. de Bot.,’
1895, p. 145.
1905.] On the Germination of Seeds of the Castor Owl Plant. 77
solution, therefore, a little ether was added, drop by drop, till a faint
turbidity was apparent.
After standing in this condition for some days, a crop of aggregates of
erystals separated out. When dissolved in water they were found to have
a specific rotatory power of about ap = +66. After inversion with a dilute
mineral acid the specific rotatory power became about ap = —18. The
solution of the crystals gave no crystalline osazone on warming with
phenylhydrazine acetate.
These reactions are fairly conclusive that the non-reducing sugar is cane-
sugar.
The reducing sugar was refractory and no method succeeded in rendering
it crystalline. It was also found impossible to separate it completely from
the cane-sugar, so that its specific reducing power could not be obtained.
Readings with the polarimeter were unsatisfactory on account of its proving
impossible to free its solutions from a yellow colouration. When the latter
were warmed with phenylhydrazine acetate they yielded a quantity of a pale
yellow osazone which analysis proved to be the osazone of a hexose. After
several recrystallisations from alcohol and from ethyl acetate the crystals
were found to have a constant melting point at 204°C. This is consistent
with the view that it is invert sugar produced from the cane-sugar with
which it is associated. It negatives the hypothesis pnt forward in the
former paper that it is derived from the glycerine of the fat, for this sugar
(glycerose), now much more completely investigated, is known to yield an
osazone melting at 130° C. to 131° C.*
The occurrence of two sugars exhibiting the characters just described
suggested a search for invertase among the constituents of the endosperm.
A good number of well germinated seeds were selected, having most of the
endosperm absorbed; the embryos were well developed, their root system
considerably branched. The endosperms were removed and ground up into
a paste, which when strained through muslin yielded 95 c.c. of an acid sap.
This was carefully neutralised and a little antiseptic added. It contained
a quantity of both reducing and non-reducing sugar, 10 c.c. of the sap
reducing 0-2 gramme of cupric oxide. Tubes were prepared containing
respectively 10 c.c. of the neutralised juice with 10 c.c. of a solution of the
non-reducing sugar from the seeds, and 10 cc. boiled juice with the same
quantity of the sugar solution, and they were digested in a water-bath at
40° C. for several hours. On titration the weight of cupric oxide reduced by
the digestion containing unboiled juice was 0°31 gramme while the other
* Fischer and Tafel, ‘ Ber. d. deut. Chem. Ges.,’ vol. 20, p. 1088; Fenton and Jackson,
‘Trans. Chem. Soc.,’ 1899.
78 Prof. J. Reynolds Green and Mr. H. Jackson. [Mar. 22,
gave the same weight as the original juice, 0°2 gramme. The treatment
with the juice had increased the original reducing power 50 per cent.,
showing the presence of invertase.
Further experiments upon the same point showed that invertase appears
in the endosperms at a very early period of germination, usually after a few
hours ; it is well established in 48 hours, and increases in amount up to the
stage at which a good root system has been established. In a series of
experiments upon its development during the germination three stages were
compared : (1) The seeds had the radicle protruding about 0:3 inch; (2) The
roots were 1 inch long and the secondary rootlets were just cracking the
primary root; (3) There was a good root system and the endosperms were
about half consumed. Extracts were made of all these and 2 c.c. of each
allowed to act on 20 c.c. of a 1 per cent. solution of cane-sugar, at 40° C. for
24 hours. They were then titrated with Fehling’s fluid, when the weights
of cupric oxide obtained were :—
(1) 0003 gramme; (2) 0:006 gramme; (3) 0-007 gramme.
These experiments lead us to the conclusion that the sugars of the
endosperms may be put down as cane-sugar and invert sugar.
The relative quantities of these two sugars during the progress of germina-
tion have been ascertained and are given in Table B. Experiments on this
point have been published by Du Sablon in the paper already referred to.
He states that he found non-reducing sugar to be slightly in excess of
reducing sugar in the resting seed and to increase more rapidly than the
latter till the radicle is about 1-5 to 2 inches long, when the reducing sugar
becomes equal in amount and, later on, preponderates considerably.
Our experiments were carried out in the following manner :—A number
of seeds were germinated in sawdust in an incubator kept at a temperature
of 22°C. In each experiment three were taken, peeled, and ground up to
a smooth paste in an agate mortar. The paste was then boiled with
a sufficient quantity of water for an hour, the extract strained off, filtered,
and divided into two. Half was warmed to 40° C. with 1 cc. of a solution
of invertase prepared from yeast, and kept at that temperature for 24 hours.
The invertase solution was ascertained to be free from sugar or other
substance capable of reducing Fehling’s fluid. The two halves of the
extract were then titrated side by side, and the weight of the cupric oxide
taken in each case. From these weights the quantities of the two sugars
were computed in the usual way. .
1905.] On the Germination of Seeds of the Castor Oil Plant. 79
Table B.
Time of orb Invert Cane-
germination Come thion Of aq eods wine sugar in sugar in
in hours. ae milligrammes. | milligrammes.
@) Resting seeds ............s00seseeeee | 11 10-7
45 Caruncle swollen ................- | 2-7 5°17
69 Little further external change...) 2°3 0)
117 Root about 0°75 inch long ...... | 6-7 19 -4
168 Root 1°35 inch long ............... 5-2 10°5
216 Roots branching .................5 | 19°65 35 °7
240 Endosperms cracking ............ | 29 -O1 35 °8
312 Good root system .............00665 | 40 °8 52 °6
|
A comparison of this Table with Table A suggests that the course of
events in which the sugars are involved proceeds upon much the same lines
as that connected with the lecithin. The cane-sugar is present in greater
quantity in the resting seeds, it gives place to invert sugar under the
influence of the invertase during the early period of germination, and
subsequently increases in amount and remains slightly in excess of the invert
sugar during the later stages when absorption is more active. This suggests
that cane-sugar is the actual reserve, and that the invert sugar represents
the form which has the greater nutritive value.
In accounting for the increase in the quantity of cane-sugar which marks
the progress of germination, it is necessary to call attention to a fact noticed
for the first time a few years ago by Mr. Biffen in the Cambridge Botanical
Laboratory. Emphasis has already been laid upon the fact that a very
vigorous metabolism in the endosperm cells is an accompaniment of germina-
tion. This was commented on by Van Tieghem* in 1877, when he found
that endosperms deprived of their embryos were capable of swelling and.
apparently starting a kind of development. In the former paper on this
subject one of us described experiments} confirmatory of Van Tieghem’s
views. Biffen has found that a considerable increase of the protoplasm
of these endosperm cells is a marked feature of the early stages of germina-
tion. The exact time at which it occurs varies somewhat, but it corresponds
fairly closely with the recommencing formation of cane-sugar. The
coincident occurrence of these two events points to a vrowth of the
protoplasm of the endosperm cells at the expense of the initial reserves,
which we have seen are undergoing conversion changes at and before this
time, and a subsequent construction of further carbohydrate reserves by
* Van Tieghem, “Sur la Digestion d’Albumen,” ‘ Comptes Rendus,’ vol. 84, p. 578.
+ Green, loc. cit., p. 389.
80 Prof. J. Reynolds Green and Mr. H. Jackson. [Mar. 22,
the protoplasm in the endosperm for the nutrition of the outlymg embryo
as its growth continues. Apart from such secretion the endosperm contains
no carbohydrate material, while the latter seems to be essential for the
maintenance of merismatic tissue. The fact that this carbohydrate substance
is cane-sugar coincides with the observation of Brown and Morris* that
cane-sugar is always present in the growing embryo of the barley-grain.
It appears to be a form of carbohydrate very suitable for serving as a
temporary reserve material, more easily utilisable than starch, and therefore
formed where the deposit of the reserve will be of very short duration,
as in the case of the embryo, and in that of the foliage-leaf, where Brown
and Morris found it at a very early period of the photosynthetic construction.
Indeed, from the results of analyses of the mixed sugars then present they
suggested that it might even be the first sugar formed.t
It may again be noted that in the case of Ricinus its formation is
accompanied or speedily followed by the secretion of invertase. The enzyme
is not present in the resting seeds, but develops in the endosperms after
exposure to a temperature of 25° C. in moist earth or sawdust for 48 hours
or less, though germinative changes are not visible so soon in the external
appearance of the seeds. The amount of the enzyme increases continuously
all the time of germination, and the invert sugar increases coincidently.
The protoplasm appears to keep up a secretion of cane-sugar and the
invertase seems to keep working on the latter, so as to supply invert
sugar at once to the protoplasm of the cells and to the young absorbing
embryo.
It will be seen from what has been said that we do not associate the
formation of this carbohydrate material during the germination directly
with the diminution in quantity of the oil which is taking place at the
same time. Our experiments lend no support to the views of Sachs that
the oil was directly transformed with either sugar or starch. The two
processes are features of a new metabolism set up in the cells as germination
becomes established. To this point we shall return later.
THE ACIDS OF THE GERMINATING SEEDS.
The question of the nature of the acid to which the reaction of the
germinating seed is due remains to be dealt with. Evidence of acidity can
* Brown and Morris, “Researches on the Germination of some of the Graminez,’
¢ Journ. Chem. Soc.,’ vol. 57 (1890), p. 518.
+ Brown and Morris, “A Contribution to the Chemistry and Physiology of Foliage
Leaves,” ‘Journ. Chem. Soc.,’ May, 1893, p. 673.
1905.| On the Germination of Seeds of the Castor Ou Plant. 81
be obtained after a seed has been exposed to warmth and moisture for
24 hours, and it becomes more and more intense for six or seven days.
While the reaction to litmus paper becomes very prominent, only very
small quantities of acid can be obtained from the seed. The expressed juice
of a parcel of germinating seeds was titrated with decinormal potash
solution, and 10 c.c. of it neutralised only 4 cc. of the alkaline solution.
We made several attempts to prepare it in quantity by experimenting upon
about a thousand seeds at once. They were germinated for a week, and
the endosperms separated from the embryos, ground and boiled in water
in a steriliser for several hours. After straining and filtering part of the
extract was distilled by the aid of steam. The distillate was practically
neutral in reaction, the merest trace of acidity coming over. The acid in
the remainder, after removal of uncoagulable proteid, was precipitated by
normal lead acetate, and the lead salt filtered off, suspended in water and
treated with a stream of sulphuretted hydrogen till the lead was all con-
verted into sulphide. The filtrate from the latter was concentrated to a
small bulk, and the precipitation and subsequent treatment repeated. The
final filtrate was concentrated to a small bulk in vacuo over sulphuric acid.
The acid residue, somewhat syrupy in consistence, was then washed
repeatedly with dry ether, which dissolved a certain quantity, leaving
behind, however, a good deal of acid which was soluble in water only.
The bulk of the latter was ascertained to be phosphoric acid. The solution
in ether was concentrated i vacuo and formed a syrupy residue. We found
it impossible to crystallise this acid or to obtain a crystallisable salt. Many
attempts were made to effect crystallisation, but in only one case was any
success obtained, and then only a few crystals on the surface of the syrup
were formed. Unfortunately, therefore, the nature of the acid has not been
ascertained.
After looking for the source of this organic acid we again find reason to
attribute it to the oil. We have already pointed out (p. 75) that the amount
of lecithin formed is not sufficient to account for the disappearance of the
whole of the oil of the seed, but that another fate awaits a considerable
quantity. It was suggested in the former paper* that the acid of the
germinating seed was derived from the oil by certain processes of oxidation,
and served as the means of its utilisation. It is extremely unlikely that this
acid is directly or indirectly connected with the sugars. We think we have
here the explanation of the gradual diminution of the oil in the early stages |
of germination, and of the development of the coincident acidity. The acid
reaction of the endosperm sets in before any change can be detected in the
* Green, loc. cit., p. 385.
VOL, LXXVII.—B, G
82 Prof. J. Reynolds Green and Mr. H. Jackson. [Mar. 22,
globoids of the aleurone grains and before any reaction for phosphoric acid is
obtainable. The probability of an oxidation of the oil taking place in the
early stages of germination has already been pointed out. This is now
‘rendered still more probable by the discovery of an oxidase in the
germinating seeds. On mixing a strained and filtered extract of the
endosperms with a solution of hydroquinone, the colour of the latter
speedily becomes pink and, later, red. The extract gives instantaneously
a blue colour with an emulsion of guaiacum, and slowly turns a solution
of pyrogallol purple. Boiling the extract destroys the power of setting up
these changes. The oxidase adheres very tenaciously to the tissue of the
endosperm, and it is very difficult to extract it completely.
Though the oxidase can be extracted and the extract found to act on such
easily oxidisable bodies as those mentioned, no attempt has succeeded in
making it oxidise ricinoleic acid outside the plant. This may, however, be
due to non-attainment of the conditions which exist in the cells of the
endosperms. ‘Though its appearance is suggestive, it has not been proved
that it plays a part in the oxidative processes of the fats, if the latter take
place. The probability of such oxidative processes is considerable, for, in
addition to the considerations just put forward, it should be remembered
that one of us has shown that the formation of the acid is dependent upon
the access of oxygen. In seeds germinated in its absence, though part of the
oil was transformed, no acid soluble in water was formed.*
The problem is complicated by the fact that the distribution of the lipase,
invertase, and oxidase of the germinating seed is practically the same.
NUTRITION OF THE EMBRYO.
The sequence of changes which has, so far, been described, suggests a
modification of the views now current as to the mode of utilisation of reserve
materials in albuminous seeds. It has been commonly held that the efforts
of the parent plant ceases with the deposition of reserve food in or near the
embryo, in such a condition as to be easily used. Possibly, also, certainly in
some cases, the parent is responsible for the provision of an enzyme to effect
the change of the reserve food into a suitable condition for absorption. The
utilisation is, however, attributed more or less fully to the embryo. In many
cases the latter secretes the enzymes itself, and in others it is the active
agent in absorption. The metabolic changes in the endosperm attributable
to the parent are held to be more or less independent of each other, and to
consist of the enzyme actions only, each enzyme fitting its appropriate food
for absorption.
* Green, loc. cit., p. 389.
1905.| On the Germination of Seeds of the Castor Oil Plant. 83
This, as we have shown, is far from being the case with Aucinus. Here we
have a series of most complex changes set up by the parent in the endosperm,
accompanied by a renewed growth and revived secretory activity of the
parent itself. The various constituents are made to act upon each other
under the influence of the protoplasm of the endosperm cells, the latter
showing a great increase in the amount of their protoplasm, while the
protoplasm initiates a complex metabolism comparable in intensity with
any which can be marked in the adult plant. It feeds itself, having
prepared the food from the reserves ; it secretes new products, which were
represented but sparingly in the original cell-contents, thus preparing a new
and completely representative food supply which it places at the disposal
of the embryo. At the same time, however, the latter plays a considerable
part in the scheme of nutrition, besides carrying out the processes of
absorption.
A study of the distribution of the enzymes of the seed shows us that the
preparation of food is not all carried out by the parent. The lipase was
stated in the earlier paper* to originate in the endosperm cells and to
continue to be developed there during the whole course of the germination.
The invertase and the oxidase appear to have a distribution similar to that
of the pase. The trypsin, however, originates in the embryo.
In the course of the researches made by Mr. Biffen, which have already
been referred to, he found that the epidermis of the young cotyledons
contained cells, occurring at short intervals, which stained quite differently
from the rest, and were full of granular contents. We prepared a large
number of cotyledons from seeds in course of germination, taking them at
an early stage when it was just possible to separate them cleanly from the
endosperm. They were then washed carefully in warm distilled water till
all organic matter was removed from their surfaces. Each cotyledon was
then cut in half along the mid-rib. One set of halves was dipped for a
moment in boiling water. The two sets were put into a solution of the
globulin of the seeds prepared by dissolving it from the seed in 10-per-cent.
solution of common salt and precipitating it by strong alcohol. The tubes
containing them were put for a few hours into an incubator at 30°C. At
* A curious misstatement of what I said on this point in my earlier paper has been
made by Connstein, Hoyer, and Wartenburg (‘Ber. d. d. Chem. Ges.,’ vol. 35 (1902),
p. 3988), and recently repeated by Vierling (‘Journ. Suisse de Chim. et Pharm.,’ vol. 42,
(1904), p. 391). Iam represented as saying that the action of the lipase is stopped by the
liberation of the acids in the endosperm. My paper contains no such statement. What
I said was that if the enzyme was set to work én vitro in the presence of dilute hydro-
chlorie acid it was rapidly destroyed. Reference to my paper will show that I regarded
the organic acids formed in the endosperm helpful and not deleterious —J. R. G.
84 Prof. J. Reynolds Green and Mr. H. Jackson. [Mar. 22,
the end of this time the uninjured epidermis ,had produced such a change
in the globulin that the solution gave a vivid reaction for tryptophane on
addition of a little chlorine water. The contents of the other tube were
unchanged. The presence of trypsin in the cotyledonary epidermis was
consequently proved. An extract of the cotyledons gave the same results.
Taking these experiments in conjunction with Mr. Biffen’s observations,
there can be little or no doubt that the special cells alluded to secrete
the trypsin.
These observations throw a light upon certain phenomena already alluded
to, which were first recorded by Van Tieghem,* and subsequently corrobo-
rated by one of us.f Van Tieghem dissected the embryos out of seeds of
Ricinus and exposed the endosperms on damp moss for some weeks to
a temperature of 25 to 30°C. After several days of this exposure he found
them growing considerably, and at the end of a month they had doubled
their dimensions. The change was caused by the enlargement and partial
separation of the constituent cells. In the interior of the cells he found the
aleurone grains to be gradually dissolving, and the oily matter to be slowly
diminishing. In the confirmatory experiments made by one of us the
changes were found to be much more rapid when pieces of the cotyledons
were left in contact with the endosperms than when the embryo was
entirely removed. No satisfactory explanation of these phenomena was.
forthcoming at the time that they were observed, but the discovery that the
tryptic enzyme is secreted by the cotyledons affords one. That a very slow
germination takes place in the complete absence of the cotyledons may be
explained by a small exudation of the enzyme from the latter before their
removal or by the endosperm-cells themselves secreting a small quantity of it
when the growth of the protoplasm is resumed during the early stages. The
diffusion of the trypsin from the cotyledons into the tissue of the endosperm
is exactly paralleled by the diffusion of diastase from the scutellum of the
barley grain, described by Brown and Morris.t
CONCLUSIONS.
The germination of the seed of Ricinus is shown by the experiments now
recorded to be associated with a remarkable activity of the cells of the
endosperm, which spring into renewed life and set up a very complex
* Van Tieghem, “Sur la Digestion d’Albumen,” ‘Comptes Rendus,’ vol. 84 (1877),
p. 578.
+ Loe. cit., p. 389.
{ Brown and Morris, “On the Germination of some of the Graminex,” ‘ Journ. Chem.
Soc.,’ vol. 57 (1890), p. 495.
1905.] On the Germination of Seeds of the Castor Ol Plant. 85
metabolism. Their protoplasm grows and takes a prominent part in these
metabolic changes, secreting enzymes, and setting up various chemical
changes in the cells partly by means of the latter and partly independently
of them. In this renewed activity the embryo also takes a share by
contributing to the enzyme-formation. The result is the production of
a great variety of nutritive material, partly the direct product of enzyme-
action, partly produced by the secretory activity of the protoplasm and
partly by the interaction of the products of the first two agents. Two
varieties of sugar, lecithin, fatty acids, and the products of their oxidation,
proteids, and the products of their digestion, including various crystalline
nitrogenous bodies, amino- and amido-compounds at least are present. In
this mass of nutritive material the embryo is plunged, and by the delicate
epidermis of its cotyledons it absorbs, probably selectively, what it needs for
its own growth. It is not easy to follow the process of absorption in detail,
on account of the metabolism accompanying growth, which is very speedily
set up in the cells of the embryo.
Analyses of the cotyledons show them to contain a varying quantity of
lecithin, amounting in some cases to 1°36 per cent. of their dry weight.
Both the sugars can be detected in them, the relative amounts, however,
varying, but cane-sugar being usually present in largest quantity.
The reaction of the sap is acid, traces of phosphoric acid being mixed with
an organic acid whose nature has not been ascertained. In fact, the
transport of the nutritive substances to the embryo seems to be much the
same in character as their transport in the tissue of the endosperm,
Probably in both cases the presence of protoplasmic threads in the various
cell-walls plays an important part in the matter; it seems at any rate
probable that this agency is necessary to explain the transport of lecithin to
the embryo. A very small quantity of lecithin can be dissolved in water or
exist as a fine emulsion. It is improbable, however, that it can be trans-
mitted through the cell-walls by dialysis alone. Dialysis no doubt plays
a large part in the absorptive processes, especially where the crystalline
substances are concerned.
The renewed metabolism in the endosperm-cells thus furnishes a mass of
nutritive material on which both the endosperm-cells and the young embryo
feed, and there seems to be no particular difference in the manner in which
they are severally nourished.
VOL, LXXVII.—B. H
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———
Oo 20 40 60 80 I00 120 140 160 I80 200 220 240 260 280 300 320 340 360
~<——_Pressuresin mm. of mercury.a——>
Fie. 1.
Haperiment 2.—Heart Muscle.
The hearts of two sheep were perfused with saline, the parts free from
visible fat were then cut out, minced and otherwise treated as above described.
The percentage of proteid was 6°28 per cent., and of ethereal extractive
1:16 per cent. The results are given in Table II and graphically in Curve 1,
fig. 2.
90
Prof. B. Moore and Dr. H. E. Roaf.
Table I1.—Temp. 40° C.
[Oct. 19,
Percentage by
Pressure of
Percentage by
SW chloroform in weight of
eines ai vapour space, chloroform
originally added, in mm. of pumped off into
mercury. vapour space.
0:05 12 62 0 :0078
O71 21°89 0 0135
0-2 47 -02 0 -0290
0'3 75 °35 0 0464
0°5 93 -87 0 0578
0°8 169 °25 0 1042
1:0 198 -49 0 1538
2°0 297 ‘11 0 1829
3°0 322 -94 0 -1988
4:0 339 88 0 -2092
5:0 334 °62 0 -2060
Percentage by
Coefficient of
weight of distribution
chloroform between
remaining in vapour space
solution. and solvent.
0 0422 1: 5°4
0 0865 1: 6°4
01710 ig 52
0 -2536 abe 159955
0 4422 ie (77
0 6958 aba Ot/
0 8462 1: 6:2
1°8171 yg (8) 8)
2 8012 1:14:1
38-7908 ato aig}
4, °7940 1 : 23°3
Experiment 3.—Lnver Tissue.
Sheep’s liver was prepared in the usual manner. The percentage of proteid
was 11:37, and of ethereal extractive 2°80. The results of the experiment are
given in Table III, and graphically in Curve 2, fig. 2.
Table III.—Temp. 40° C..
Percentage by
weight of
chloroform
originally
introduced.
ARwWNrOOoCOO
SSSSCSHAWNE
Pressure of
Percentage by
Percentage by
chloroform in weight of weight of
vapour space, chloroform chloroform
in mm. of pumped off into remaining in
mercury. vapour space. solution.
9-63 0 0059 0 0941
20°85 0 :0128 01872
32 84 0 -0202 0 :2798
56 “41 0 0347 0 4653
85 58 0 0527 0 7473
108 °21 0 -0666 0 9334
161 -26 0 0993 1 -9007
199 -29 01227 2 8773
239 -75 0 1476 3 8524
267 -80 0 1649 4°8351
Coefficient of
distribution
between
vapour space
and solvent.
:15°9
:15°4
:13°9
:13°4
:14°2
: 14:0
: 19-2
:23°5
: 26-1
: 293
A ee ee pe
1905.] On certain Properties of Solutions of Chloroform, etc. 91
U
U
Oy)
gy /
v
}
solution. ———>
20 40 60 80 100 120 140 160 I80 200 220 240 260 280 300 320 340 360
~——Pressures in mm. of mercury.—#_
<«—— Percentages of chloroform by weight in
Fic. 2.
92 Prof. B. Moore and Dr. H. E. Roaf. [ Oct. 19,
B.—Experiments on the Relationship between Vapour-pressure and Concen-
tration of Chloroform in Emulsions in Saline of the Ethereal Extractives
(Lipoids) of Serum and Brain Tissue.
These experiments were devised with the object of testing whether the
alterations in the relationships between vapour-pressure of the anesthetic and
its concentration, as compared with water and saline, found in the case of
serum and the tissues, were due entirely to lipoids or ethereal extractives
contained in these fluids, or whether part of the effect was due to action of
the proteids. The results shown in the curves of figs. 1 and 3, clearly show
that a great deal of the action is due to the proteid.
That a certain amount of the anesthetic will be taken up by the lipoid in
a physical fashion there can be no doubt on account of the high solubility of
chloroform and other anesthetics in such lipoid substances. But we hold
that the portion of anesthetic so taken up and held by the lipoid is passive
and not active, and that it is the other portion taken up by the proteid (the
existence of which figs. 1 and 3 demonstrate) which is active in paralysing
protoplasmic activity and producing anesthesia.
It is a matter of common experience that the greater the amount of fatty
tissue in a subject undergoing anesthetisation, the greater the amount of
anesthetic required. The portion of anesthetic which is absorbed by the
lipoid is imprisoned as far as purposes of anzsthetisation are concerned, and
so much the more anesthetic must be given in order to raise the pressure of
anesthetic sufficiently and cause combination between cell-protoplasm and
anesthetic with resulting anesthetisation.
The ethereal extractives (lipoids) were obtained by the following method :—
The proteid of the serum or brain tissue was completely precipitated by
addition of excess of absolute alcohol, and the precipitate was separated from
the alcohol. The precipitate was thoroughly extracted with ether. The
absolute alcohol solution was evaporated to dryness and the residue also
thoroughly extracted with ether. The two ethereal extracts were united and
the ether evaporated off. The total ethereal extractive was weighed and then
made up into a fine emulsion by shaking with normal saline (0°75 per
cent.). The volume of the emulsion was made equal to that of the serum
originally taken, and in the case of the brain tissue the concentration of the
emulsion of the ethereal extract was made equal to the amount of lipoid
directly determined in the sheep’s brain. For comparison with the results in
the case of the ethereal extractive emulsion of serum, the results in the case
of serum from our former paper are given, and the comparison is shown in
the two curves of fig. 3. In the case of the brain tissue and the emulsion of
the lipoids of brain tissue of equal concentration, the results are shown
alongside in the two curves of fig. 1.
1905.] On certain Properties of Solutions of Chloroform, etc. 93
Separation or Coagulation of the Lipoid Emulsions by Chloroform and other
Substances.
An interesting physical effect is seen as the amount of chloroform added to
the emulsion of lipoids in saline is increased. Ata certain stage, dependent
upon the richness of the emulsion in lipoid, a complete separation of the
lipoid, in a butter-like mass, is obtained, leaving the saline practically free
from lipoid. The phenomenon suggests a resemblance to the similar precipi-
tation of proteid observed under like conditions, but there is this difference,
that in the case of proteid, when the amount of anesthetic is sufficient, the
precipitate is permanently altered, being coagulated and rendered insoluble in
water or saline, while the lipoid is only physically thrown out, and can be
re-dissolved in ether and again made into an emulsion with saline. This
phenomenon of physical aggregation of the lipoid by the anesthetic is the
more remarkable because the emulsions are exceedingly permanent and
remain unaltered for days. The permanency is probably due to lecithin, and
the emulsion under the microscope shows small bodies, which are not in
most cases spheres, but show the appearance of bi-concave discs of varying
size, many being no larger than mammalian blood-corpuscles. The physical
cause for the production of such discs is at present unknown to us, but the
matter is being further investigated.
In the case of the emulsion of brain tissue (containing 4°07 per cent. of
lipoid) coagulation or separation of the emulsion occurs in the cold when
about 2 per cent. of chloroform has been added, and the coagulation occurs
much earlier at body temperature. The coagulum or separated lipoid forms
a jelly-like mass, which later separates into a thin whitish fluid and a butter-
like mass. These emulsions can also be coagulated or separated by solutions
of neutral salts, alcohol, benzol, xylol, and other organic fluids.
The precipitation may be due to a lowering of surface tension in the
emulsion, and presents an interesting analogy with the precipitation of
proteids and other colloids from solution by neutral salts, chloroform, and
other organic substances which act as anesthetics. It may be noted in this
connection that froth on serum disappears when chloroform is dropped into it,
and we have noticed the absence of frothing on stirring on the chloroform
side of our densimeter as compared with the control side.
Expervment 4.—Ethereal Extract of Serum.
The extract was obtained as described, the percentage of ethereal extractive
(lipoid) in the emulsion in saline as introduced into the densimeter was 0-206.
The results are given in Table IV, and shown graphically in Curve 2, fig. 3, °
where they are contrasted with the results given by entire serum.
94 Prof. B. Moore and Dr. H. E. Roaf. [Oct. 19,
Table IV.—Temp. 40° C.
Percentage by Pressure of Percentage by Percentage by Coefficient of
weight of chloroform in weight of weight of distribution
chloroform vapour space, chloroform chloroform between
originally in mm. of pumped off into remaining in vapour space
introduced. mercury. vapour space. solution. and solvent.
0-1 38 -60 0 -0238 0 -0762 1:3°2
0:2 77°41 0 0476 01524 1:3°2
0:3 108 °32 0 0667 0 2333 1:3°5
0°5 177 “71 0 °1094 0 °3906 1:3°6
0°8 262 ‘60 0°1617 0 6383 1:3°9
1°0 309 :27 0 +1904 0 8096 1:4°3
1:2 351 ‘90 0 °2105 0 '9895 1:4°7
Poel a ae
be f
/
/
<— Percentages of chloroform by weight in solution. —>
fo} 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360
~«— Pressures.in mm. of mercury. —>
Fig. 3.
1905.] On certain Properties of Solutions of Chloroform, etc. 95
Experiment 5.— Ethereal Extract of Sheep’s Brain.
The emulsion, prepared as before, contained 4:067 per cent. of ethereal
extractives. The results are given in Table V, and are contrasted graphically
with the results obtained from the emulsion of the entire brain in fig. 1,
Curve 1, brain tissue; Curve 2, ethereal extractives.
Table V.cTemp. 40° C.
Percentage by Pressure of Percentage by Percentage by Coefficient of
weight of chloroform in weight of weight of distribution
chloroform vapour space, chloroform chloroform between
originally in mm. of pumped off into remaining in vapour space
introduced. mercury. vapour space. solution. and solvent.
0-103 32 °44, 0 0199 0 0831 1:4°2
0-206 52°15 0 :0320 0 -1740 1:5°4
0 309 62 12 0 :0381 0 2709 iL faa
0°515 107 ‘11 0 0656 0 4494 1:6°9
0-820 145 °31 0 0890 0 -7310 1:8:2
1-040 190 °22 0°1161 0 9234 1:7°9
The experiment could not be carried beyond this point because the lipoid
separated out at 40° C. at all higher concentrations in chloroform.
C.—Relative Solubilities of a Series of Anesthetics in Water and Serwm
respectively, and the Effects of Addition of such Anesthetics wpon the Serum.
The method of determining the solubility of the anesthetic consisted in
weighing out known amounts of the anesthetic into water and serum
respectively, stoppering the flasks, placing on the mechanical shaker for some
hours, and then determining by direct observation that concentration in each
case at which the anesthetic ceased to be dissolved.
The anesthetics so tested were chloroform, ethyl ether, ethyl acetate, amyl
aleohol, amyl acetate, benzol, and xylol. The results obtained were as
follows :—
Chloroform.
Solubility in water at 13° C.......
4 serum _,,
0:95 per cent.
400 ,,
In water, 0°8 per cent., all dissolved ; 0-9 per cent., all dissolved ; 1 per cent.,
not dissolved completely ; estimated solubility, 0°95 per cent. In serum,
3 per cent., all dissolved ; 3°5 per cent., all dissolved ; 4 per cent., all dissolved
save a few small globules; estimated solubility, almost 4 per cent.
96 Prof. B. Moore and Dr. H. E. Roaf. [(@et.” 19%
Ethyl Ether.
Solubility in water at 15° C....... 8 per cent.
pA Serum (ene nl eee TM
At 10 per cent. and over in the serum there was a slight opalescence, and
the fluid began to grow gelatinous ; 11 per cent., completely dissolved ; 12 per
cent. not dissolved ; and at higher percentages two phases separated, the layer
on top forming a clear, thin jelly.
Ethyl Acetate.
Solubility in water at 15° C....... 79 per cent.
pe SN 5 as baoae 10
At 8 per cent. complete solution of the ethyl acetate and commencing
precipitation of the proteid occurred. Solutions of 9 and of 10 per cent.
strengths also dissolved completely. Separation into two phases occurred at
12 per cent., the upper layer being viscid.
Amyl Alcohol.
Solubility in water at 15° C....... 2-4 per cent.
5 Serum), yen) Wes 8 ms or over.
At 3 per cent. complete solution of anesthetic with commencing precipita-
tion of proteid. This precipitation gradually increased with concentrations of
3°5 and 4 per cent. up to 8 per cent., when a thick cream was formed. A
separation into two phases could not be observed at any stage.
Amyl Acetate.
Solubility in water at 15° C....... 0:25 per cent.
e Serum... ut eee 15 Bs
At 1 per cent., complete solution of anesthetic with precipitation of proteid ;
at 1°5 per cent., all dissolved except a few minute globules.
Benzol.
Solubility in water at 15° C....... 0-15 per cent.
a Seruin > 48) ee 06 a
At 0°5 per cent., commencing precipitation and opalescence; 0°6 per cent.
completely dissolved; 0-7 per cent., not all dissolved.
Xylol.
Solubility in water at 15° C....... 0:016 per cent.
\. BEL} ih Rye Cee 0:2 i
1905.] On certain Properties of Solutions of Chloroform, etc. 97
At 01 per cent., complete solution, cloudy from proteid precipitation ;
0:2 per cent., all dissolved; 0°3 per cent., not all dissolved.
In all cases the solubility in serum is higher than in water, and it is clear
from the results that a similar association between the anesthetic and the
proteid of the serum occurs throughout the series. The amount of ethereal
extractive in the different samples of serum used for the determinations
varied between 0:24 and 0°36 per cent., thus showing that the increased
solubilities found could not be due to solution of the anesthetic in lipoid or
ethereal extractives present in the serum.
D. Changes in Depression of the Freezing Point of Water, Saline, and Serum
respectively caused by the addition of Chloroform.
These experiments were undertaken to elucidate if possible the state in
which the chloroform existed in the proteid solutions.
On account
of the chloroform being volatile, the apparatus had to be completely closed,
and the stirring accomplished by electro-magnet in the usual way. The
solution was placed in a straight tube, which was almost completely filled, so
that no correction was required for escape of chloroform into the air space
above the fluid.
The changes in depression of freezing point are small, but in every case
the results are comparable and concordant. In the table (Table V1) the
percentages of chloroform are given by weight, and the figures are corrected
to allow for the degree of supercooling.
The determinations were made with Beckmann’s apparatus.
Table VI.—Changes in A for Amounts of Chloroform added to Various
Solutions indicated at the Heads of the separate Columns.
Saline g Sor Sonam
Distilled water solution Serum Serum cha 2 te eiae
plus 0°6 per (0-75 per | plus 0°6 per | plus 1:0 per ot nes an on ap “9
cent. cent.) plus cent. cent. be ey rs As is ae
chloroform. oad Reon chloroform. | chloroform. Pi peareeralichionat ae |chicsotecn
0-061 0-081 0-060 0-101 0°135 0-138 0 142
0 ‘075 0-075 0 :062 0 -086 0-141 _— —
0:077 0:072 0:072 0-104 _ = =
0-099 0 082 — — — = =
Average 0:078 0078 | 0 ‘065 | 0 ‘097 0188 0°138 0 °142
It is noticeable from the table (1) that the lowering of freezing point for
chloroform in serum is less than the lowering due to the same amount of
98 Prof. B. Moore and Dr. H. E. Roaf. [Oet. 195
chloroform in water or saline; (2) that the lowering of freezing point in the
case of serum goes on increasing far beyond the point of maximum solubility
of chloroform in water or saline, but that the increased lowering is not
strictly proportional to the extra amount of chloroform added, but progres-
sively less; and (3) above 2 per cent. further addition of chloroform has no
more effect upon the freezing point. The latter fact points to a removal of
the chloroform by the proteid corresponding to the steep rise in the vapour-
pressure curves at|high concentrations.
E. Changes in Electrical Conductivity in Saline and Serum produced by the
addition of Chloroform.
The electrical conductivities were determined by the method of Kohlrausch,
at the temperatures of 0°, 15°, and 40°C. In the case of saline (0°75 per
cent.) and different samples of serum, both before and after addition of
definite weighed amounts of chloroform. In the case of the saline the
amount of chloroform added was 0°6 per cent. by weight, and in the case of
serum amounts of 0°6, 1, 2, and 3 per cent. by weight were added in succes-
sive experiments. On account of variations in the different samples of serum
there are considerable fluctuations in the results, but certain facts are
observable. In Table VII the alterations in the value of K (the specific
conductivity, multiplied by 10”) are shown, which result from the addition of
the amounts by weight of chloroform shown at the head of each column. It
is noticeable that the conductivity is reduced without exception in all cases
where chloroform is added to normal saline, due to a diminution of the
ionisation of the sodium chloride by the chloroform and alteration of ionic
velocity. But when chloroform is added to serum there is, in many cases, an
actual increase in conductivity, and in other cases the diminution is less than
occurs with the corresponding amount of chloroform added to normal saline.
This indicates that there is a tendency for inorganic salts to be set free from
the proteid and add to the conductivity. This is only seen as an actual
increase where it outbalances the action above mentioned of the chloroform
in reducing conductivity in pure saline solution.
The table shows that the amount of increase in conductivity varies with
the temperature and the amount of chloroform added. Thus at 0° C. the
diminution in conductivity is the same in saline and serum for 0°6 per cent.
of chloroform, but when 1 per cent. of chloroform has been added, the
diminution becomes converted into an increase; again, at higher percentages,
a diminution is seen, as if all the electrolyte possible had been detached from
the proteid at the lower concentrations, and the diminution was now that due
to the effect of the additional chloroform upon the saline in solution (fig. 4).
1905.| On certain Properties of Solutions of Chloroform, etc. 99
Table VII.—Changes in K x 10? for amounts of Chloroform added to Serum
and Saline.
Saline solution
(0°75 per cent.) | Serum plus Serum plus Serum plus Serum plus
Temperature. plus 0°6 0°6 per cent. | 1:0 percent. | 2:0 percent. | 3:0 per cent.
per cent. chloroform. chloroform. chloroform. chloroform.
chloroform.
© Oh
(0) — 0:0140 — 0:0267 + 0:0015 — 0:0275 — 0:0176
0 — 0:0204 — 0:0077 + 0:°0160 — 0:0139 — 0:0275
Average .... — 0°0172 | — 0:0172 | + 0:0087 ge 0 0207 — 0:0225
15 — 0:0360 + 0:0390 + 0:0440 + 0:0140 — 0:0060
15 — 00020 + 0:0460 + 0:0890 — 0:0050 — 0:0130
15 — 00361 + 0-0032 — 0:0146 — 0:0255 — 0:0336
15 — 0:0200 — 0-0092 — 0:0044 — 0°0162 — 0:0323
Average . — 0:0235 | + 0:0197 + 0:0285 | — 0:0082 | — 0:0212
40 — 0:0270 — 0:0240 — 0:0320 — 0:0410 ==
40 — 0-0300 — 0:0270 — 0-0880 — 0:0690 ae
40 — 0:0410 — 0:0140 — 0:0470 — 0:0650 —
40 — 00399 — 0:0170 — 0:0331 — 0:0577 — 0:0674
40 — 0:0314 — 0:0061 — 0:0312 — 0:0669 — 0:0488
Average ‘ — 00339 — 0:0176 | — 00463 | — 0:0599 | — 0:0581
OaG i
O-o1
OD bret rve cle ate 2 sd
O05 3:0
Percentages of ehigroronm by weigce in solution.
Change in specific conductivity x 10%.
Fic. 4.—Changes in Electrical Conductivity on addition of Chloroform to Saline (Curve 1)
and Serum (Curve 2) at 0° C. Between the first two points the curves are identical.
The effects are most marked at 15° C.; here, instead of the diminution occur-
ring on addition of chloroform to pure saline, there is found an increase in
conductivity in the case of serum both for addition of 0-6 per cent. and 1 per
100 Prof. B. Moore and Dr. H. E. Roaf. [Oct. 19,
cent., which as before becomes converted into a diminution for 2 per cent.
and 3 per cent. (fig. 5). At 40° C. there is no actual increase at any concen-
Q
fe)
=)
5 Chee in specific conductivity x 107.
fo)
fe)
nN
0°03
Rorooe ee of aoe by oat in Sanne
Fie. 5.—Changes in Electrical Conductivity on addition of Chloroform to Saline (Curve 1)
and Serum (Curve 2) at 18° C.
@
°
-
2
fe)
iy
Change in specific conductivity x 10*.
°
c}
ur
0:06 O5 TO 2:0 30
Percentages of chloroform ly weight in solution.
Fic. 6.—Changes in Electrical Conductivity on addition of Chloroform to Saline (Curve 1)
and Serum (Curve 2) at 40° C.
tration, but the fall in conductivity is less for serum than saline at 0°6 per
cent., and this fall is increased at the higher concentrations in chloroform
(fig. 6).
1905.] On certain Properties of Solutions of Chloroform, etc. 101
Taking the experiments as a whole, there is evidence of two opposing
factors partially masking each other, the first being due to a setting free of
electrolyte from the proteid by the added chloroform increasing the con-
ductivity, and the second the well-known action of a non-electrolyte in
diminishing the conductivity, which is seen clearly in the case of the pure
saline plus chloroform.
Summary and Conclusions.
The experiments recorded in the present communication support the
conclusion drawn in our previous paper that anesthetics form unstable
compounds or aggregates with the proteids of the tissue cells, and that
anesthesia is due to a paralysis of the chemical activities of the protoplasm
as a result of the formation of such aggregations.
The comparative experiments with ethereal extracts demonstrate that the
action is upon the cell proteids and not upon the lipoids.
The compounds or aggregations so formed are unstable, and remained
formed only so long as the pressure of the anesthetic in the blood is
maintained.
The results of our experiments may be summarised as follows :—
(1) The solubility of all anesthetics experimented with is higher in serum
than in water.
(2) At a certain concentration, definite for each anesthetic, there occurs
opalescence and commencing precipitation of proteid.
(3) At equal concentration of chloroform in water or saline on the one
hand, and serum, hemoglobin, or the tissues (brain, heart, muscle, and liver)
on the other, the vapour-pressure is always higher in the former than in the
latter.
(4) The curve connecting vapour-pressure and concentration is, in the case
of water and saline, a straight line; while in the case of serum, hemoglobin.
and the tissue proteids it is a curve showing association, especially at the
higher concentrations.
(5) Comparative determinations of vapour-pressure and concentration, in
serum and brain tissue and in ethereal extracts of these equal in concentration
of lipoid, show that the proteid of the tissue combines with the anesthetic.
(6) Determinations of the effects of addition of chloroform upon the lower-
ing of freezing point confirm the results obtained by the vapour-pressure and
solubility determinations.
(7) Determinations of the changes in electrical conductivity caused by
addition of chloroform indicate that accompanying the combination of the
anesthetic with the proteid there takes place a splitting off of electrolytes.
VOL. LXXVIIL—B. I
102 Prof. Moore, Dr. Roaf, and Mr. Whitley. [ Oct. 9,
(8) When the lipoids, extracted from serum or tissues by ether are made
up into an emulsion with normal saline, many of the lipoids take the form of
bi-concave discs.
(9) The lipoid emulsions are very permanent, but separate on the addition
of anesthetics or neutral salts, in similar fashion to colloidal solutions.
On the Effects of Alkalies and Acids, and of Alkaline and Acid
Salts, upon Growth and Cell Division in the Fertilized Eggs
of Kchinus esculentus.—A Study in Relationship to the
Causation of Malignant Disease.
By Benyamin Moore, M.A., D.Sc., Johnston Professor of Bio-Chemistry,
University of Liverpool; Herbert E. Roar, M.D. Toronto, Johnston
Colonial Fellow, University of Liverpool; and Epwarp WuirtLey, M.A.
Oxon.
(Communicated by Professor W. A. Herdman, F.R.S. Received October 9,—
Read November 23, 1905.)
The results of observations previously made in the Bio-chemical Laboratory
of the University of Liverpool have shown that free hydrochloric acid is
absent from the gastric contents, or greatly reduced, in nearly all cases of
malignant disease, no matter where the malignant growth happens to be situated.
In the paper describing these observations it was pointed out that the
most probable cause of this absence of the free hydrochloric acid was an
increased alkalinity of the blood-plasma, as a result of which the hydrogen
ion concentration in the plasma was so far reduced that the oxyntic cells
were no longer able to separate an acid secretion from it.*
It seemed to us, therefore, desirable to test the effects of alterations in the
concentration of hydrogen and hydroxyl ions respectively, upon the growth
and cell division in some organism in which cell division was proceeding
rapidly, and which could be easily subjected directly to changes in acidity or
alkalinity of the medium in which it was living.
We selected for this purpose the fertilized eggs of a species of sea-urchin
(Echinus esculentus), because at the season of the year at which our experi-
ments were carried out (April, 1905) the ripe eggs can readily be obtained
from the gonads of the female, and be fertilized by mixing with the sperm
similarly obtained from a ripe male. Hence from the single cell stage
* “Roy. Soc. Proc.,’ B, vol. 76, p. 138, 1905.
1905.] Liffects of Alkalies, etc., on Eggs of Echinus. 103
onward the rate of growth and any irregularities in cell division can be
observed, and the effects of the addition of alkali or acid, or alkaline or acid
salt, can be compared under exactly similar conditions, and contrasted with
a control grown alongside in unaltered sea-water.
The experiments were made, by kind permission of Professor W. A
Herdman, F.R.S., at the Marine Biological Station, Port Erin, Isle of Man,
during the month of April, 1905.
J. Loeb,* in 1898, reported experiments on the influence of acids and
alkalies on the development of the larve of sea-urchins, showing that the
addition of acids to the sea-water delayed the development, and finally, at a
certain concentration of hydrogen ions, inhibited the development com-
pletely. With the addition of alkalies the development during the first
12 hours was not at all hastened, or hastened to a hardly appreciable extent.
On the second day, however, and sometimes also even on the third day,
the eggs that developed in sea-water which had been made alkaline were
occasionally, but not always, in advance of eggs of the same brood which had
been raised in normal sea-water.
At the time of the publication of this paper, Loeb held the current view
that sea-water had an alkaline reaction, and accordingly drew the conclusion
that, for developmental processes, it is necessary to have an alkaline reaction
or, in other words, a higher concentration of hydroxyl ions than exists in
distilled water.
At a later date Loeb,f however, came to the conclusion that normal sea-
water must be regarded as a practically neutral solution, and hence ascribed
the favouring action of alkalies upon cell-division and growth to the
neutralizing action of the alkali added upon acid products formed by the cells
in the process of growth which would otherwise cause an accumulation of
hydrogen ions and arrest the development.
This opinion is based by Loeb upon the results of the observations of
Friedenthal,t Fraenkel§ and Farkasj| which indicate that blood-plasma
possesses no higher concentration in hydroxyl ions than distilled water ; upon
observations made by Dr. Cottrell at Loeb’s suggestion upon the sea-water of
the Bay of San Francisco by means of hydrogen electrodes, which showed
* © Arch, f. Entwickelungsmechanik d. Organismen,’ 1898, vol. 7, p. 631.
+ ‘Arch. f. d. ges. Physiol.,’ 1903, vol. 99, p. 637; zb¢d., 1904, vol. 101, p. 340; zbed.,
1904, vol. 108, p. 506 ; ‘Univ. of California Publications, Physiology,’ 1908—4, vol. 1,
pp. 39, 139.
t ‘Arch. f. Anat. u. Physiol., Physiol. Abth.,’ 1903, p. 550.
§ ‘Arch. f. d. ges. Physiol.,’ vol. 96, 1903, p. 601.
|| ‘Arch. f. d. ges. Physiol.,’ vol. 98, 1903, p. 551; see also Hiber, ‘Arch. f. d. ges.
Physiol.,’ vol. 99, 1903, p. 572.
it 7
104 Prof. Moore, Dr. Roaf, and Mr. Whitley. [Oct. 9,
that the concentration of hydrogen ions was greater by one power of ten than
in distilled water ; and upon the observations of Loeb himself, (1) that normal
sea-water is practically neutral to phenol-phthaléin, and (2) that addition of
sodium bicarbonate or of di-sodic phosphate, which from their neutral
reaction to phenol-phthaléin are regarded by Loeb as neutral salts, produced
the same favouring effect upon cell-division and reproduction as the caustic
alkalies.
A solution of the mixed phosphates or carbonates in which there is an
approximate balance between the concentration of hydrogen and hydroxyl
ions such that these concentrations are nearly equal, cannot, however, be
regarded as neutral in the same sense as distilled water is neutral; or as being
acid or alkaline in the same sense as a solution containing only free acid or
free alkali can be regarded as being acid or alkaline.
Nor will such a solution of phosphates and carbonates as is present in blood-
plasma or sea-water have a similar action upon living cells to either distilled
water or a neutral solution of such salts as sodium chloride of equal osmotic
concentration.
Such solutions form a peculiar type of their own, which possess the
property of behaving either as an acid or as a base. Such solutions can
behave in this manner to any substances possessing weak acid or basic
properties brought into the same solution, and it is for this reason that such
solutions behave entirely differently to distilled water in their associating or
dissociating action upon coloured indicators, and so indicate at the same time
an acid reaction to one indicator and an alkaline to another.
A quantity of acid or of alkali can be added to the solution, which would
cause an enormous variation in the relative concentration of hydrogen and
hydroxyl ions if added to distilled water, without altering to anything like
the same extent the relative concentrations of these two ions.
Therefore blood-plasma, and to a less extent sea-water, possess, on account
of the mixed phosphates and carbonates which they contain, a steadying action
upon variations in the concentrations of the hydrogen and hydroxyl ions.
When acid or alkali is added to the plasma, instead of there occurring a
corresponding swing in the concentration of the hydrogen and hydroxy] ions,
there takes place an alteration in the equilibrium of the ions of the phosphates
and carbonates, which neutralizes, in great part, the hydrogen or hydroxyl
ions added, and prevents the plasma becoming markedly acid or alkaline.
Without such a controlling action the life of the cells would be rendered
impossible, for, as our experiments show, the living cell is most sensitive to
even small variations in either hydrogen or hydroxy] ion.
This powerful action of alterations in concentration of hydrogen or hydroxyl
1905. | Liffects of Alkalies, Go, on Lggs of Echinus. 105
ion, arises from the fact that the constituents of the protoplasm behave like
very weak acids or bases, and are affected by variations in hydrogen or
hydroxyl ions in a similar manner to coloured indicators. Very small
variations in ionic concentrations compared to those found in even dilute
solutions of free acid or alkali, will hence cause the constituents of the cell to
become almost completely associated or dissociated, and prevent the chemical
reactions from occurring which are necessary for the metabolism and life of
the cell.
This is shown in our experiments by the very small amounts of either acid
or alkali which suffice to kill the cell. Within the limits at which life is still
possible, but at which the concentrations of the two ions are varied from the
normal, profound alterations occur both in the rate of growth, and in the
type of the cell divisions.
In the case of malignant growths similar variations in hydrogen and
hydroxyl ions occur as shown by the absence of the acid in the gastric juice.
It is from this point of view that we have studied the effects upon growth
and cell-division of the addition of acids or alkalies in small amounts to the
medium in which such processes are taking place.
The problem before us is that of the effects upon the cell of variations in
the hydrogen and hydroxyl ion concentration, and for the purposes of such an
enquiry it is not neccessary to know what are the actual concentrations of the
two ions in normal plasma, nor to discuss which of these two magnitudes is
the greater.
If alkali be added to plasma or to sea-water the concentration in hydrogen
ions will fall and that in hydroxyl ions will rise, and conversely on adding
acid a reverse change will take place. The experiments we have made show
the results of such changes upon cell growth and nuclear division.
A number of other observers have drawn attention to irregularities in cell
growth and nuclear division induced by the action of foreign chemical
substances upon the dividing cells.
Thus, O. and R. Hertwig,* and also Galleottit have shown that pathological
mitoses with irregular division in number and amount of the chromosomes
may be induced by such substances as quinine, chloral, nicotine, anti-pyrine,
cocaine, and potassium iodide.
The derangements in division so produced closely resemble the pathological
divisions described in cancer cells by Klebs,t Hansemann,§ and Galleotti|
* *Jenaische Zeitsch. f. Naturwissenschaft,’ vol. 20, 1887, pp. 120, 477.
+ ‘Beit. z. path. Anat. u. z. allgem. Path.,’ vol. 14, 1893, p. 288.
t ‘ Die allgem. Pathologie,’ Fischer, Jena, 1899, Part 2, pp. 528, et seg.
§ ‘ Virchow’s Archiv,’ vol. 119, 1890, p. 299 ; vol. 123, 1891, p. 356.
|| Loe. cit.
106 Prof. Moore, Dr. Roaf, and Mr. Whitley. [Oct. 9,
Hansemann recognizes in such abnormal mitoses two general groups, (1)
asymmetrical mitoses, in which the chromosomes are unequally ditributed to
the daughter cells, and (2) multipolar mitoses, in which the number of
centrosomes is more than two, and more than one spindle is formed.
Hansemann and Galleotti find in this asymmetry of mitoses an explanation
of the well-known fact that in cancer cells many of the nuclei are especially
rich in chromatin (hyperchromatic cells) while others are abnormally poor
(hypochromatic cells). According to Galleotti, the asymmetrical mitoses
which may be artificially produced in the epithelial cells of salamanders
by treatment with dilute solutions of anti-pyrine, cocaine or quinine are
exactly like those seen in carcinoma.
In these experiments, the drugs used for inducing pathological mitoses are
not such as can occur in carcinoma. A variation of the concentration in
hydregen and hydroxyl ions can, however, occur in the plasma, and, in fact,
the observations on the absence of acid in the gastric secretion in cases of
- carcinoma make it probable that such variations do occur.
It is hence of interest that in our experiments detailed below, in which the
concentration of hydrogen and hydroxyl ions was artificially varied, we have
found that with increased alkalinity, at a point just short of that at which
cell growth was stopped, such pathological mitoses do occur.
We have observed both the asymmetrical mitoses with unequal distribution
of chromosomes and only two centrosomes, and the multipolar mitoses with
three or more centrosomes. Also, as the alkali was increased above the
normal of sea-water, a marked tendency to irregularity in size and shape of
the resulting cells was observed similar to that seen in cancer cells.
As the amount of alkali was increased, there occurred also a shortening of
the dividing chromatin rods, similar to that seen in most maturation divisions,
until the rods became in some cases converted into rounded dots. In a
certain percentage of the divisions, the number of chromosomes was reduced.
The number of chromosomes is exceedingly difficult to count with certainty,
but the reduction in many cases amounted approximately to one-half the
normal,
EHaperimental Methods.
We have investigated in our experiments the effects of the following
alkalies and acids, and alkaline and acid salts :—
Sodium hydrate, potassium hydrate, calcium hydrate, ammonia, hydro-
chloric acid, acetic acid, carbonic acid, sodium carbonate, sodium bicarbonate
mono-sodium phosphate (NaH,PO,), and di-sodium phosphate (Na,HPO,).
The stock solutions of the acids and caustic alkalies were made in distilled
water, titrated and standardized to normal strength, and from these deci-
1905. | Effects of Alkalies, etc., on Eggs of Echinus. 107
normal solutions in distilled water were prepared. The carbonates and mono-
' sodie phosphate were made up in decimolecular strength, and the di-sodic
phosphate in 54, molecular strength, on account of its lower solubility. A
saturated solution of calcium hydrate was prepared in distilled water
decanted from the lime, and standardized against decinormal acid, and this
standardized solution was used in small measured amounts for addition to
the sea-water containing the samples of eggs as in the other cases.
Similarly, in the case of the carbonic acid, a comparatively strong solution of
carbonic acid was prepared by passing the gas through sea-water ; this solution
was standardized at once with decinormal alkali, using phenol-phthaléin as
indicator, and immediately added in appropriate small measured quantities to
the various measured sea-water and egg-mixtures.
It may be pointed out that there was no appreciable variations in osmotic
pressure of the sea-water caused by the addition of the reagent solutions,
because the volume added was small in comparison with the volume of sea-
water and egg-mixture to which it was added; further the decinormal solu-
tions added, though hypotonic, do not lie very far below the molecular
concentration of the sea-water. If the decinormal solutions had been
made in sea-water instead of distilled water, an equal or greater amount of
change in osmotic pressure would have resulted on account of their being
hypertonic, and also precipitation of constituents of the sea-water would in
certain cases have occurred. The change in osmotic pressure was, however,
in all cases quite a negligible quantity.
The experiments involved the use of a large number of vessels on account
of the long series of mixtures of sea-water and fertilized eggs with their
varying amounts of added chemicals of different kinds, and we had no*
anticipated this heavy demand when starting upon our expedition. Hence,
we had to make use of such materials as we could find in the Marine
Biological Station, or obtain at Port Erin. The earlier experiments were
made in well washed out glass jam-pots such as are used for collecting fresh
marine specimens, but later we found ordinary plain tumblers of the usual
size most convenient.
The amount of surface for aeration compared to the volume of fluid was the
same in each case throughout each series of experiments, and henee the
results obtained are strictly comparable with one another.
The method of procedure in starting an experiment was as follows :—The
shells of a number of Echini were cut open circularly so as to expose the
uninjured gonads, until a ripe male and a ripe female had been obtained as
shown by examining under the microscope, and ascertaining that the
spermatozoa were active, and the eggs of mature size and well formed.
108 Prot. Moore, Dr. Roaf, and Mr. Whitley. — [Oct 9,
Usually two gonads from the female, and about half to one gonad from the
male were taken, each in a separate tumbler, gently rubbed up with a small
quantity of sea-water, and then separated from débris by filtering through a
coarse piece of muslin. The eggs were somewhat diluted with sea-water, a
quantity of sperm added, and a drop of the mixture taken out and examined
with the microscope until it was ascertained that the eges had developed their
fertilization membranes.
A number of tumblers corresponding to the number of controls and the
total of the various dilutions of the different chemicals to be tested for their
effects upon the growth of the eggs had previously been arranged and
numbered. The mixture of fertilized eggs was now diluted to a larger volume
with sea-water so as to afford 200 cc. for each tumbler, and this volume
was measured out into each of the tumblers. As rapidly as possible the
desired amount of each chemical was added in each case, and the time of
starting noted. The progress of development was observed and noted a few
hours after starting the experiment, again the following morning, and so on.
In a few cases instead of diluting the egg-mixture after fertilization to a
large volume, the proper amount of sea-water was measured out into each
tumbler, then an equal volume of the egg-mixture was added to each, followed
by the desired amount of the chemical solution.
For the purpose of examining the progress of development a dip was
taken out by means of a small pipette into a, watch-glass and examined
under a low power of the microscope. The state of development of the
growing embryo was noted especially with regard to relative rate of growth
in presence of the various strengths of the different alkaline and acid
solutions. The number of cells was counted in the earlier stages, or the number
of cells in an optical section of the circumference of the blastule in the
later stages; the commencement of ciliary motion was noted, and the stages
in the development of the gastula in those cases where the larve developed
so far. Also any irregularities in shape and size of cells in the different cases
were noted.
In certain cases, the progress of any change in chemical reaction was
noted by adding indicators in parallel experiments carried out alongside.
Interesting results as to the action of the indicators themselves were so
obtained. It was found that the reaction to “di-methyl” did not change
throughout the experiment, but the reaction to phenol-phthaléin, which was
faintly alkaline even in normal sea-water at the commencement of the
experiment, slowly changed towards the acid side. As it was found, however,
that phenol-phthaléin even in very small amounts inhibited and caused
irregularities in cell-division, the method was adopted of testing quantita-
1905. | Effects of Alkales, etc., on Eggs of Echinus. 109
tively by titration the reaction to both phenol-phthaléin and “ di-methyl” at
the end of each experiment after growth had stopped. It was found that
even where alkali had been added at the commencement of the experiment
the reaction to phenol-phthaléin had become slightly acid at the end in most
experiments. Since the amount of alkalinity to “di-methyl” had not altered
throughout the experiment, but gave in all cases very approximately the
alkalinity of sea-water plus or minus the alkali or acid artificially added, it
follows that the change in reaction to phenol-phthaléin must be due to a
very weak acid given off during the development of the embryos. Since
carbon-dioxide produces exactly the same effects, and respiration must occur
in the process of development, it is almost certain that the change in
reaction must be due to production of carbonic acid. On account of this
natural production of carbonic acid, the results of the first twenty-four
hours’ growth are the most valuable, because here the alkaline reaction to
phenol-phthaléin persisted, and the production of carbonic acid was small.
After preliminary experiments had determined the range at which the
developing embryos were definitely affected by the different chemicals, a few
final experiments were made in which only two or three concentrations of
each chemical were included lying at about the proper range, and the last
such experiment was interrupted when the more advanced sets of embryos
had reached the morula or early blastula stage.* The embryos were fixed in
Flemming’s and Hermann’s fluids, embedded and cut in paraffin, and stained
for nuclear division by Heidenhain’s iron-alum and hematoxylin staining,
following Flemming’s description. The drawings illustrating the paper were
made from this series of preparations, with the exception of fig. 18, which
was drawn from the fresh growing cells.
Reaction of Sea-Water of Port Erin Bay—One hundred cubic centimetres
of sea-water was taken and titrated against decinormal caustic soda solution,
(a) with di- methyl- amido- azo- benzol as indicator, and (0) with phenol-
phthaléin as indicator. With the “di-methyl” in a first trial 2°35 c.c. were
required, in a second 2°37, therefore the alkalinity was = 0:00236 normal.
With the phenol-phthaléin 0:24 c.c. and 0:22 c.c. were required, alkalinity
= 0°00023 normal.
* After cutting sections, it was found that many of the cells described in Table XI
(pp. 123, 124) in the fresh condition as morule really possessed in section a small central
cavity, and so are termed blastule in the descriptions of the drawings. The organisms
described as blastule in the tables showed in the fresh condition an outer layer of cells.
110 Prof. Moore, Dr. Roaf, and Mr. Whitley. [Oet. 9,
EXPERIMENTS WITH
Table IL—
Experiment No. 1,
| : ate
| Amount of sodium Amount of added alkali Observations of condition
No hydrate in decinormal in the solution, ex-
| ; solution, added to pressed as a fraction of
200 c.c. of sea-water. normal strength. 4 hrs. 17 h. 50 m.
| 1 Control — Four-celled stage | Blastule ............
|
2 0-2 c.c. 0 -0001 Four-celled stage | Blastule ............
3 | 0-4 ,, 0 0002 Four-celled stage | Blastule ............
4 | OSes 0 :0004 Four-, six-, and| Blastule. Further
eight - celled| advanced in cell-
stages. Irregu-| division than con-
lar divisions trol
5 1‘4 ,, 0 :0007 Four-celled stage.) Blastule. Further
| Irregular divi-| developed than
| sion of cells control
6 2°0 ,, 0 0010 Majority two-| Blastule. Further
celled. A few} developed than
four-celled. control
* In order to distinguish throughout between different sets of eggs, each lot of fertilized
It is to be noted in this experiment that there is a distinct favourimg action of the added
stages of No. 6, but not in sufficient amount to stop growth (see Experiment 2), and as the alkali
Also Nos. 4, 5 and 6 remain alive and develop further than Nos. 1, 2 and 3.
No. 5 was, at the end of the period 91 h. 50 m., divided into two portions of 100 c.c. each,
it. The subsequent development of 50, which had the alkali added, was much more rapid than
1905. | Effects of Alkalies, etc., on Eggs of Echinus. 111
Caustic ALKALIES.
Sodium Hydrate.
Brood* of Eggs No. 1.
of embryos, at interval after start of experiment given at head of each column.
24h.50m.| 29h.15m. | 41h. 45m. | 70h. 30m, 91h. 50 m. 114 h.
Blastule. A| Blastule. All| Blastule. All| Commencing gas-} All dead, and —
few moving| in active | in active | trule. Nearly| degenerating
motion motion all dead
Blastulez. A} Blastule. All| Blastule. All | Commencing gas-| All dead, and _—
few moving| in active | in active | trule. Nearly) degenerating
motion motion all dead
Blastule. A/| Blastule. All} Blastule. All | Commencing gas- — —
few moving | in active | in active | trule. All dead
motion motion
Blastule. A} Blastule. All| Blastule. All| Well -developed| Late gastrule. | —
few moving} in active | in active | gastrule. All| Very active
motion motion active (glass broken
by accident)
Blastule. Blastule. All| Blastule. All} Well - developed} Late gastrule.| Forming
Motionless | in active | in active | gastrule. All| Not so far de-| plutei
motion motion active veloped as No. 4
Blastule. Blastule. All| Blastule and | Well - developed} Late gastrule. —
Motionless | in active | some gastru-| gastrule. All| (Formalin
motion le. All in| active added by mis-
active motion | take)
eggs is numbered as belonging to the same brood.
alkali in the earlier stages, especially observable in No. 4; the alkali is in excess in the earlier
becomes diminished by the CO, given off in growth, No. 6 increases and passes the control.
of which the first (5a) was left unaltered, while the second had 0°5 c.c. of N/10 NaOH added to
that of 5a, and the embryos remained alive much longer.
| Oct. 9,
Prof. Moore, Dr. Roaf, and Mr. Whitley.
112
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113
Liffects of Alkahes, etc., on Eggs of Echinus.
1905.]
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1905. | Effects of Alkalies, etc., on Eggs of Echinus. 115
Table VimAmmonium Hydrate.
Experiment 1, Brood No. 3.
| i t of | Amount of f :
;| Amount or | added alkali | Observations of embryos at stated times after commencement
SE LERTNOATAES What in the of experiment,
decinormal | sation
q 7 2
No ee ae expressed aS} __
380), g f a fraction
| ane of normal
sea-water. strength. 2 hrs. 16 h. 30 m.
1 Control | — | Division into two cells com- | Blastule. Twenty to twenty-
mencing four cells in circumference
2) 05 ce 000025 |Sameascontrol .............. Blastule. Twenty cells in cir-
| cumference. A few morule
3 0°75 ,, 000037 | Same as control ............... Same as No. 2
4 TL O) 5 0 0005 Same as control ............... Same as No. 2
5 oS y; 0:00075 | Same as control ............... Half morule and half blastule
|
6 2:0 0-001 No division .........00.e.00008 Same as No. 5
Experiment 2, Brood No. 6.
In this series blastulee formed with smaller
number of cells in embryo.
Amount of
Amount of added alkali | Observations of embryos at stated times after commencement
ry
RUTTEN in the of experiment.
decinormal Tati
solution ee
eerie expressed as
200 c.c. of a fraction
sea-water. | 0%, noxmal 18 h. 30 m, 42 hrs.
strength.
Control — Four-celled, many morule | All stages to early blastule
and a few early blastule
i ce 0 -0005 Two-, three- and four-celled, | Morule, at all stages
and morule
2 op 0-001 Many single - celled, and | Harly and late morula. Ex-
stages up to early morule trusion of cell contents from
cells
By, 0 -0015 Nearly all single cells ......... No further development. Ex-
trusion of cell contents
116 Prof. Moore, Dr. Roaf, and Mr. Whitley. (Oct...
Table VIL.—Calcium Hydrate.
Experiment 1, Brood No. 5. The lime-water added required for neutrali-
sation of 50 cc., in one case 25:25 cc. of N/10 HCl, and in a second
trial 25°28 c.c.; this lies so near 25 cc. that the solution was takem
as N/20.
| Amount of
Amount of | jaded alkali | Observations of embryos at stated times after commencement
calcium in the of experiment.
hydrate in Tuti
No. | N/20 solu- rae ag
tion, added | ©*Prpssty as -
Aaa ae) fraction
sea-water. eee 18 hrs. 42 hrs.
il Control — | Well-developed blastule...... No further development. Com-
mencing degeneration
2 1 ce. 0:00025 | Blastule clearly more ad-| Late blastule, one or two
vanced than control, and| moving
showing more cell-division
3 2s 0 -0005 Same as No, 2 ..........2.0005 Same as No. 2, but more in
motion
4 4 ,, 0-001 Well-developed blastule, but} Blastule, but motionless
some deformed in shape
5 @ 5 0:0015 Morule, and occasional small | Blastule, but not so far
blastulee developed as No. 4
6 8 ,, 0-002 Nearly all single-celled; but | Nearly all single-celled. Those
occasional two-, three- or| showing development are
five-celled group chiefly early irregular morule.
Single cells show extended
layer.
7 10 ,, 0. 0025 Single cells in many cases} No development; single cells
| pear-shaped showing extended layer
A second experiment gave similar results.
inus. ial 7¢
etc., on Eggs of Ech
2
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1905.]
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118 Prof. Moore, Dr. Roaf, and Mr. Whitley. [ Oct. 9,
Table VIII.—Acetie Acid.
Experiment 1, Brood No. 6.
Amount of
Amount of | “Jaded acid | Observations of embryos at stated times after commencement
acetic acid in th of experiment.
in N/10 in the P
No solution, ee
added to | °XPressed as
200 c.c. of a fraction
sea-water. O: Dome 18 h. 30 m. 42 hrs.
strength.
1 Control — Many morule and early| All stages to well-formed
blastule. Some four-celled | blastulse
groups
2 0°5 c.c. 0:00025 | Morule, occasional early | All stages to late morule.
blastula. Not so far ad-|} Division irregular
vanced as control
3 10) 55 0 -0005 Morule only. Irregular | Same as No. 2
division
4A 20 ,, 0 -0010 Early stages and irregular | Karly morule, and some late
morule. Many cells not| morule. Cells breaking up
divided
5 3:0 ,, 0 :0015 None beyond six- and eight- | Chiefly early morule, and
yf § :
celled stages earlier stages. Irregular
division in earlier stages
6 4:0 ,, 0 002 All single-celled ............... No stages beyond eight-celled.
Irregular division
"/ 5:0 ,, 0:0025 | Allsingle-celled ............... All single-celled
IIS)
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1905.]
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1905. | Fiffects of Alkalies, etc., on Eggs of Echinus. 125
Summary of Results on Rapidity of Growth.
The following conclusions may be drawn from the observations on the
developing eges in the living condition recorded in Tables I to XI, which
are also illustrated by figs. 1 to 17, drawn from sections of the eggs developed
in the experiment described in Table XI. The sections were drawn under a
low magnification (61 diam.) with the Zeiss Zeichen-ocular. The eggs
shown in the drawings had all developed, for the same period (8 hours) in
the different media specified, from the same brood of eggs, and the eggs were
subsequently fixed and stained as described above.
1, The extreme limits of variation of hydrogen and hydroxyl ion within
which growth is possible are very narrow. Addition of 0:0015 M. of caustic
alkali (see figs. 3 and 7) on the one hand or of 0:001 M. of acid (see fig. 4)
practically stopping all cell-division.
2. A slight inerease in alkalinity favours growth and cell-division and at
the same time tends to produce irregularity in size and shape of the
resulting cells (contrast fig. 1 with figs. 2, 6, 8, 9, 10, 11, 12, 15, 16, 18).
3. In the case of small additions of acids no such favouring action is
observable, but from the beginning cell-division and growth are inhibited
(see Tables).
4. Increase in alkali above the optimum amount leads to increased and
irregular nuclear division unaccompanied by complete cell-division. As a
result the cells become multi-nucleated. The cells also become excessively
irregular in size and shape (see figs. 3, 7, 8, 10, 15, 16, 17, 18).
5. On the other hand increased acidity leads in many cases to action upon
the chromatin of the nuclei, so that in the sections the nuclei stain faintly
and are comparatively few in number, and there is no proliferation of nuclei
in the undivided cells, similar to that seen in the case of alkali (see figs. 4, 5,
13, 14).
6. The primary factors affecting the rate of growth appear to be the
variations in concentration of hydroxyl and hydrogen ions. Thus all
the caustic alkalies are of approximately equal power and there is little
or no action of the Kation (see Tables and contrast figs. 2 and 3 with
figs. 6 and 7). But in the case of the phosphates of the alkalies where the
hydrogen and hydroxyl ion concentrations are comparatively low, there
appears in addition to be a specific factor. See the marked action of
00025 M. NaH,PO, in practically stopping cell division, while 00050 M. of
NazHPO, has a favouring action (contrast figs. 13 and 14 with figs. 15,
Ho; 17).
126 Prof. Moore, Dr. Roaf, and Mr. Whitley. [Oct. 9,
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Fie. 11.—Growth of eggs in sea-water, Fic. 12.—Growth of eggs in sea-water,
+0:0015 M. sodium carbonate. +0:003 M. sodium carbonate.
128 Prof. Moore, Dr. Roaf, and Mr. Whitley. [Oct. 9,
Fig. 13.—Growth of eggs in sea-water, Fic. 14.—Growth of eggs in sea-water,
+0:0025 M. mono-sodium phosphate. +0:0035 M. mono-sodium phosphate.
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Fic. 15.—Growth of eggs in sea-water, Fie. 16.—Growth of eggs in sea-water,
+0'0025 M. di-sodium phosphate. +0:005 M. di-sodium phosphate.
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Fic. 17.—Growth of eggs in sea-water, Fic. 18.—Drawings of cells in fresh condition
+0:0075 M. di-sodium phosphate. developed in alkaline media, showing
irregularities in size and shape of cells.
1905. | Effects of Alkalies, etc., on Eggs of Echinus. 129
Fie. 19.—Undivided egg in sea-water, Fic. 20.—Undivided egg in sea-water,
+0°0025 M. di-sodium phosphate, +0:001 M. potassium hydrate, showing
showing two dividing nuclei. several multipolar mitoses.
Fig. 21.—Cell in blastula in sea-water, +0°001 M. potassium hydrate,
showing multipolar mitosis.
Fic. 22.—Cell in morula in sea-water, +0:001 M. potassium hydrate, showing
asymmetrical mitosis—unequal number of chromosomes.
130 Prof. Moore, Dr. Roaf, and Mr. Whitley. [Oct. 9,
Fie. 23.—Cell in blastula in normal sea-water.
Fic. 24.—Cell in blastula, in sea-water, 4.0:0025 M. di-sodium phosphate, showing
rod chromosomes, unreduced in number
Fic. 25.—Cell in morula, in sea-water, +0:005 M. di-sodium phosphate, showing
partial shortening of rod chromosomes.
1905. | Effects of Alkalies, etc., on Eggs of Echinus. 131
Fic. 26.—Cell in morula, in sea-water, +0°0075 M. di-sodium phosphate, showing
shortening of rod chromosomes to dots, and reduction in number.
Fic. 27.—Two cells in blastula, in sea-water, +0°005 M. di-sodium phosphate, showing
partial shortening and reduction of chromosomes and protrusion of spindles, as if
polar bodies were being formed.
Fig. 28,—Egg in two-celled stage in sea-water, +0°0015 M. potassium hydrate, showing
shortening of dividing chromosomes to dots and reduction in number.
132 Prof. Moore, Dr. Roaf, and Mr. Whitley. [Oct. 9,
Fic. 29.—Cell in blastula, in sea-water, +0°0025 M. di-sodium phosphate.
(See text, p. 134.)
Pathological Nuclear Divisions.
The irregularity of growth in sea-water to which alkalies or alkaline salts
have been added is accompanied by characteristic irregularities in the mitotic
divisions, which are illustrated in figs. 19 to 30.*
Multiple Nuclec—tThe divisions in the cytoplasm occur less frequently than
in the nuclei, so that the cells become multi-nucleated (see figs. 2, 6, 8, 9, 10,
15, 19, 20). This occurs both in undivided and divided eggs. In many
cases two or more such active dividing nuclei are found in the same cell
(fig. 19).
Multipolar Mitosis—This occurs both in single-celled eggs and in the later
stages (see figs. 20 and 21). The chromosomes are unequally distributed to
the different spindles.
Asymmetrical Mitosis——This frequently occurs in the various alkaline
solutions, and gives rise to unequal nuclei. The cause of the unequal number
of chromosomes appears to be that some of the chromosomes are carried to
* The figs. 19 to 30 were drawn with the Zeiss Zeichen-ocular, the finer details being
filled in as realistically as possible, under a Leitz =; oil immersion objective. The
magnification measured by the stage micrometer was 790 diameters.
1905. | Liffects of Alkalies, etc., on Eggs of Echinus. 133
one pole without previous division. (See fig. 22, and lower spindle in
fig. 19.)
Changes in Chromosomes and Reduction in Number.—The short, rod-shaped
chromosomes of the organism which we have been examining (Hchinus
esculentus) do not show typically in its normal maturation or maiotic divisions
the appearances described by Farmer and Moore* and others in maiotic
division, and by Farmer, Moore, and Walkert in cancer.
According to Bryce,t the main changes in Zchinus esculentus during
maturation consist in a marked shortening and thickening of the chromosomes
with reduction in number. Each chromosome divides into two short curved
rods with spherical enlarged ends, giving rise to an appearance similar to
tetrads. But at no time are there any rings or other irregular figures, as
described in so many other organisms, and there is no true tetrad formation.
R. Hertwig§ has observed in the case of Echinus a formation of bodies
described as resembling tetrads, as a result of addition of dilute poisons.
In our spe¢imens of eggs grown in dilute alkali, we have observed
appearances in many cases similar to those shown in the drawings illustrating
Bryce’s paper.
As the amount of added alkali is increased, there occurs both a shortening
and thickening of the chromatin rods and a reduction in their number. In
some cases the appearance of rods is lost entirely, and the chromatin becomes
arranged in minute masses resembling tetrads. These changes are illustrated
in figs. 24, 25, 26, 27, and 28. Thus in the three strengths of di-sodium
phosphate solution a shortening of the chromosomes in fig. 25 is seen as
compared with fig. 24, and in the strongest solution, fig. 26, the rods are reduced
to dots and the number is decreased to approximately one-half. In fig. 28
some of the chromosomes present a tetrad-like appearance. These changes,
while frequent in occurrence, are not seen in all the dividing cells in any
strength of solution, but occur in increased number in the stronger solution.
A peculiar appearance is represented in fig. 27, in which a decided protrusion
of one pole of the spindle in two adjacent cells of a blastula was observed
beyond the cell margin, as if a polar body were being formed.
Although it is difficult to count the chromosomes accurately, the reduction
in number is obvious on contrasting the weaker and stronger solutions.
In a fair number of cells, especially in the di-sodium phosphate solutions, a
peculiar arrangement of the chromatin is observed, which is illustrated in
* Q. J. M.S.) vol. 48, 1905, p. 489.
+ ‘Roy. Soc. Proc.,’ vol. 72, 1903, p. 499.
t ‘Q. J. M.S8., vol. 46, 1903, p. 177.
§ ‘Sitz. Ber. Ges. Morph. und Phy.,’ Miinchen, 1895. Quoted from Wilson, “The Cell
in Development and Inheritance,” 1904, p. 256.
VOL, LXXVII.—B.
134 Prof. Moore, Dr. Roaf, and Mr. Whitley. [Oct. 9,
fig. 29. The entire chromatin of the cell is seen to be arranged in two groups
of circles at a distance apart corresponding to the usual distance of the
centrosomes of a spindle, and in some cases, as in fig. 29, the remains of
achromatic fibres are indistinctly seen between the two groups of circles.
The cytoplasm surrounding each group of circles is distinctly lighter and
freer from granules than the rest of the cytoplasm. In structure each circle
consists of a thin ring of chromatin showing distinct thickenings, about four
in number, arranged approximately about equidistant around the circum-
ference. The interior of the circle is clear, and both the outer and inner |
border are clearly marked. In some cases this arrangement of the chromatin
in circles is also seen where there is only one group of circles as in
fig. 30.
Discussion of Results and Summary.
Our attention was attracted to the study of the effects of small variations
in reaction upon the growth of cells from the bio-chemical point of view,
as a result of the observation that in malignant disease no hydrochloric acid
is in general secreted by the gastric glands, no matter where the malignant
growth is situated, which pointed to an increased alkalinity of the plasma.
In the course of our investigations upon the rate of growth of the cell,
when microscopic examination was made of the cells in the fresh condition,
we were struck by the marked irregularities in size and shape of the developing
cells in the alkaline media illustrated in fig. 18, which is drawn from cells
in the fresh solution developing in sea-water, to which di-sodium phosphate
has been added, and also by the marked tendencies to nuclear proliferation.
This led us secondarily to a cytological investigaticn of the cells when fixed
and stained to show nuclear division, as a result of which we have found the
irregular forms of mitosis described in the text. These atypical divisions,
which have been produced by variations in the medium similar to those
which occur in the blood in cases of malignant disease, closely resemble the
pathological divisions seen in the growths of malignant disease.
The results of our experiments and their relationship to the processes
in malignant growths may be summarised as follows :—
1. In nearly all cases of malignant disease the secretion of hydrochloric
acid by the gastric glands is stopped or greatly reduced, and this effect is not
due to local conditions in the stomach, since it occurs wherever the growth
is situated ; but is due to a change in the distribution of salts in the plasma
whereby the alkalinity is increased or the concentration in hydrogen ions
diminished.
2. Addition of small amounts of alkalies or alkaline salts, such as
di-sodium phosphate, to the medium in which cells are growing and dividing,
1905. | Effects of Alkalies, etc., on Eggs of Echinus. 135
causes at first an increase in rate of growthand division, but as the amount
is increased, there appears a marked tendency to irregularity in size and
shape of the resulting cells. Nuclear division becomes in advance of
cytoplasmic division, so that the cells become multi-nucleated. As the alkali
is further increased, both cell division and nuclear division are stopped.
3. Accompaning the increased stimulus to nuclear division given by the
dilute alkali, there are seen many of the atypical forms of mitosis described
in malignant growths. The variations from the normal illustrated in the
drawings are: (1) multiple nuclei in the same cell in active division;
(2) multipolar mitosis, occurring both in the single cell stage, and later
in the development of the organism; (3) asymmetrical mitosis, leading to
unequal distribution of chromosomes to the two daughter cells; (4) reduc-
tion in length of the chromosomes as the strength of alkali is increased until
the chromosomes appear as rounded dots, and accompanying the reduction in
length there is also a reduction in number to about one-half the normal;
(5) in certain cases the chromatin becomes arranged in circles, each of which
shows a number of thickenings. The circles are arranged in groups in the
cell, and appear to represent a stage in the anaphase, the groups being placed
at about the usual distance apart of the centrosomes, and traces of the
achromatic fibres being occasionally visible.
4. No such increased growth or stimulus to nuclear division is given by
varying the normal reaction of the medium in the opposite direction, by the
addition of equal small amounts of acid. From the beginning the minutest
amount of added acid has an inhibitory effect upon growth and nuclear
division. There is no nuclear proliferation as the amcunt of acid is increased,
and at a very slight amount of increased acidity all attempt at cell-division
ceases. In the fixed and nuclear stained preparations cell-division figures
are absent, and in the resting nuclei the staining power of the chromatin is
decreased, so that the nuclei present a washed-out appearance contrasted with
the normal nuclei or those of organisms grown in dilute alkaline solution.
5. The extreme limits at which life and cell-division are possible lie close
together, indicating that the cell is very sensitive to even slight changes in
the concentration of hydrogen and hydroxyl ion concentration. Thus the
addition of so little as 0:0015 M. of either alkali or acid to sea-water
practically stops all growth. On account of the presence of phosphates and
carbonates in the sea-water the change in hydrogen and hydroxyl ions
caused by such additions cannot be large.
[Note added October 24.—Since the paper was written we have had an
opportunity through the kindness of Messrs. J. E. 8. Moore and C. E Walker
L 2
136 Lifects of Alkales, etc., on Eggs of Echinus.
of the Liverpool Cancer Research, of examining the appearances represented in
figs. 29 and 30 under a specially high magnification of about 3000 diameters.
The magnification was obtained with a 10-inch tube, 3 mm. Zeiss
apochromatic objective, 27 compensating ocular, and with monochromatic
green illumination. Messrs. Moore and Walker have pointed out to us that
these bodies, which appear as described in the text when seen with an
ordinary ;),-inch oil immersion, are, when seen with the 3000 diameter
magnification, really spheroidal bodies consisting of an inner mass or vesicle
which scarcely stains at all, over which ramify filaments of deeply staining
chromatin. Further examination with this magnification also demonstrates
that the chromosomes represented as dots in fig. 28 are, in reality, also
vesicular chromosomes. Even with the ;4-inch oil immersion we had been
able to see a well-marked black line around the periphery of each dot, but
had been unable to determine whether or not this was merely an optical
effect. The 3000 diameter magnification, however, clearly shows that the
structure is the same as that of the larger masses in figs. 29 and 30.
It is interesting that these minute chromosome masses occur upon the
spindle in exactly similar fashion to the normal rod-shaped chromosomes, and
that wherever they occur there is a diminution in the number of chromo-
somes. In many cases an occurrence of both rod chromosomes and spheroidal
chromosomes is observable upon the same spindle as if conversion had only
partly taken effect.
Where conversion of the entire number of chromosomes into the spheroidal
variety is seen upon the spindle, the number of such chromosomes is usually
approximately half the normal number, as in fig. 28. But when they are
found after separation has taken place, as in figs. 29 and 30, the number
is very variable, and as the number diminishes there is a corresponding
increase in size in the individual chromatic vesicles.
We are informed by Messrs. Moore and Walker that bodies presenting a
similar appearance are seen in malignant growths, and also under other
pathological conditions, and in certain normal tissues.
The question of the relationship, if any, of the diminution in number when
such spheroidal chromosomes are formed, to the reductions occurring in
normal maiotic division, and in cancer cells, we prefer to leave at the present
entirely open, since we have no evidence that the diminution in number is
effected by the same means; and we merely point out the diminution of the
number of chromosomes upon the ‘spindle as a result of increasing the
alkalinity of the medium, as an interesting and suggestive fact.]
137
A Note on the Effect of Acid, Alkali, and certain Indicators im
Arresting or otherwise Influencing the Development of the
Eggs of Pleuronectes platessa and Echinus esculentus.
By Epwarp Wuit ey, M.A. (Oxon.).
(Communicated by Professor W. A. Herdman, F.R.S. Received November 14,
—Read November 23, 1905.)
I. Errect of AcID AND ALKALI ON THE EGGS OF PLEURONECTES.
While working last spring at the Port Erin Biological Station on the
effect of acids and alkalies upon the development of Echinus eggs, it was
suggested to me that it might be interesting to try the general effect of
similar solutions upon some other type of organism.
For this purpose the eggs of the Plaice (Plewronectes platessa) were selected,
as they were to be obtained in abundance from the fish-hatchery attached to
the station, but, as time pressed, it was only found possible to experiment
with one acid and one alkali, and decinormal solutions of hydrochloric acid
and sodium hydrate were accordingly used.
In a pond attached to the hatchery numbers of plaice are kept in the
Spawning season, and the surface of the pond is skimmed each morning for
the purpose of collecting the eggs, which are then placed in the batching
apparatus. Three batches of these eggs of different ages were taken for
experimentation—those freshly skimmed from the pond, those which had
been removed two days before and had remained since in the hatching
apparatus, and those taken 10 days before and similarly treated. Some few
eggs always escape the net in the process of skimming, and are taken in the
catch of later days, so that, when 10 days old eggs, for instance, are spoken of,
what is meant is that none can be younger than that, although some few may
be slightly vlder. Such as showed obvious differences in age from the
majority of each batch were removed.
The eggs were treated in a similar way to that employed in a research
carried out at the same time on Echinus eggs,* namely, they were placed in
small batches in a number of tumblers, each containing 200 c.c. of sea-
water, to which measured amounts of the decinormal solutions of acid or
alkali were added. A summary of previous work on the effects of acid and
alkaline solutions upon development will be found in the paper above
referred to.
* See preceding paper.
138 Mr. E. Whitley. Effect of Acid, etc., on [Nov. 14,
The accelerating effect of small quantities of alkali on growth observed by
Loeb in the case of Tubularia,* and the eggs of Arbacia,t and in those
of Echinus, in the research mentioned above, was not noticed, but attention
may be drawn to the very powerful results of even a small disturbance of the
chemical equilibrium, which can be seen from Table I, where it will be
observed that, after six days, 4 c.c. of decinormal acid or alkali in 200 cc. of
sea-water (v.¢., a five-hundredth normal solution) produces a death-rate among
fresh eggs of 75 and 44 per cent. respectively, against only 5 per cent. in the
Control.
In the experiments above alluded to with the eggs of Hehinus esculentus, it
was found that acids and acid salts above a very small concentration pro-
duced more deadly effects than corresponding quantities of alkalies and
alkaline salts, and this was also found to be the case with Pleuronectes,
and can be well seen in the same table (1) for strengths of 2°5 c.c. and
upwards, especially with the younger eggs.
Probably three factors enter into the explanation :—
(1) The fact that part of the alkali added is immediately thrown out
of solution as insoluble hydrates or carbonates.
(2) Alkali is constantly being used up to neutralise the acid products
of metabolism—chiefly COs.
(3) According to Loeb, the presence of weak alkali assists the absorption of
oxygen by the organism. If this be the case, the eggs in the acid solutions
not being able to absorb oxygen so readily as the others, might probably
be less resistant to the action of the reagent.
The tables seem also to show conclusively that the younger eggs are
far more sensitive to the action of the acid or alkali, and generally to
the influences of their environment than are the older eggs, or newly-hatched
larvee, which are, indeed, extraordinarily resistant. Thus, on referring again
to Table I, it will be seen that a very large percentage of the older eggs
survived 7, and even 11, days’ treatment, whereas, of the fresh eggs, over
25 per cent. in the Control, and a much larger number in all the other cases
were dead within 10 days. In this table the effect only of very small
quantities of acid or alkali is given (five-hundredth normal and under), but
experimentation with somewhat larger amounts gave the same result as shown
in Table II. Thus, it needed only 6 c.c. of decinormal sodium hydrate to kill
all the fresh eggs in four days, but more than 8 c.c. to kill all those of two
days old in the same time, while, by the end of that period, 30 c.c. of alkali
* ‘Univ. of California Publications, Physiol.,’ vol. 1, 1904, p. 137, and ‘ Arch. f. gesant.
Physiol.,’ vol. 101, 1904, p. 340.
+ ‘Arch, f. Entwickelungsmechanik,’ vol. 7, p. 631.
1905.| the Development of Eggs of Plaice and Echinus. 139
which was the greatest strength employed, had killed only 65 per cent. of the
10-days-old eggs. Acids, above a small concentration, had a remarkably
stronger effect—in one day even 10-days-old eggs being killed by 10 c.c., and
the two other batches by 6 e.c.
When working with the larger quantities of alkalies, it was very difficult
to tell exactly when an egg was dead. In the first experiments the point
was taken at which the egg began to become opaque, but, as soon as the
percentage of alkali present exceeds a very small amount, precipitation of
calcium and magnesium hydrates takes place, which renders observation
of such a change in the transparency of the egg difficult. Loeb, in his
experiments on Fundulus,* finds that the precipitate itself acts injuriously
upon the eggs, and that, if this be filtered off, the eggs will live and
develop in much stronger solutions than they would otherwise do. The process
of filtering off the precipitate before placing the eggs in the solutions was not
tried in the course of these experiments, they having been carried out
previously to the reading of Loeb’s paper.
Hitherto, it has always been considered that the young larva, on first
hatching from the egg, enters upon the most critical stages in its career,
and is at that time most susceptible to external influences, but the experi-
ments here carried out appear to show a resistance steadily increasing with
age, and that, even after the rupture of the egg-capsule, the young larva is, at
all events, no more susceptible than just before that event. It may be
suggested that this steadily increasing resistance is due to the gradual
development of the epidermal cells, which would form a protection to the
young embryo more or less impervious to the surrounding solution.
Tables III, IV, and V give the actual experimental data on which Table I
is founded, and of which it is a résumé. Table VI is introduced with the
object of laying emphasis on the statement as to the resisting powers of the
older eggs. It shows the percentage of these eggs which succeeded in
hatching in spite of the very unnatural conditions (stagnant water, possible
overcrowding, etc.) in which they were placed. Incidentally may be noticed
the very deadly effect of “di-methyl,’ to which attention is now to be
drawn.
I]. Errect oF INDICATORS ON PLEURONECTES AND ECHINUS.
The effects which di-methyl-amido-azo-benzol and phenol-phthalein produce
upon living organisms, as illustrated by the eggs of Pleuronectes and Echinus
esculentus, were accidentally discovered in the course of these experiments.
These indicators were originally added to the contents of some of the
* © Arch. f. Entwickelungsmechanik,’ vol. 7, p. 631.
140 Mr. E. Whitley. Effect of Acid, ete., on [Nov. 14,
tumblers, to show any changes in reaction that might take place during
growth. When it was observed that they had a specific action upon the
eggs, a series of experiments was undertaken with them, the results of which
are shown in Tables VII—X. In all the experiments, except those recorded
in Table X, two drops of the indicator were added to 200 cc. of sea-water, or
sea-water plus varying quantities of alkali, in a tumbler. Table X records
the results of varying the amount of phenol-phthalein employed. It will be
seen that, although the indicators were made up in alcoholic solution, the
amount of alcohol added in each case to 200 cc. of liquid, was quite
insufficient to materially influence the result.
The experiments without indicators, recorded in Tables VIII and IX,
are included for purposes of comparison.
It will be observed from the figures obtained that dimethyl is very
deadly to the eggs of Pleuronectes and phenol-phthalein innocuous, while the
opposite holds good with Echinus, the dimethyl having, if anything, a
favouring effect on growth, and the phenol-phthalein being very injurious.
Before killing, phenol-phthalein appears to be very effective in producing
irregular divisions. The dimethyl was readily absorbed as such by both
the organisms, staining them a deep yellow, so there can be no question
as to its having thoroughly penetrated the tissues.
So far as can be ascertained, this specific action of indicators has not been
noted before, and no explanation can be given of the fact of the different
indicators affecting the two organisms in exactly opposite ways. Tadpoles in
tap-water, to which the same, and even much larger amounts of these
indicators had been added, appeared to be totally unaffected by either.
It might be of interest to repeat the experiments with other organisms,
and with other organic compounds not known already to act as poisons.
Summary.
(1) The amount of variation from the normal concentration of hydrogen
and hydroxyl ions in sea-water which the eggs of Pleuronectes will tolerate
is very small.
(2) A disturbance of the equilibrium towards the acid side is much more
fatal than the opposite.
(3) A progressive development of resistance to an unfavourable action of
the environment takes place in proportion to the age of the eggs.
(4) Phenol-phthalein is deadly to the eggs of Hchinus esculentus, but
harmless to those of Pleuronectes, while dimethyl quickly kills the latter,
and appears, if anything, to have a favourable influence upon the develop-
ment of the former.
1905.] the Development of Eggs of Plaice and Echinus. 141
My best thanks, in conclusion, are due to Professor Herdman, Ren:
to whose kindness I am indebted for the material for these experiments,
and for permission to work at the station, and to Professor Moore, for his
kind and valuable criticism and assistance throughout.
Table I—Comparison of Percentages of Deaths in Fresh Eggs. Eggs of
2 days old and eggs of 10 days old, with varying quantities of deci-
normal NaOH and HCl.
After After = After | After After After
6 days. | 7days. | 7 days. | 10 days. | 11 days. | 11 days.
Fresh 2days | 10days | Fresh | 2 days 10 days
eggs. old. old) ‘eggs, |, old: old.
| |
Control, 200 ¢.c. sea- 5:0 0 2°0 25:0 0 2°4
water. (9 days)
1-0 c.c. decinormal NaOH 4°3 0) 4-2 |
1°5 » "2 9°6 = 0 29 |
2°0 » 5 25 0 ) 0-9 |
2°5 p es 18-2 2°6 3°0 31°8 2°6
3°0 20 BS 27°3 12°7 2°5 31°8 14°5 2°5
4:0 op a 44°0 250 9°4 48 -0 48 2 26 °9
(10 days)
1-0 c.c. decinormal HCl...) 10-0 3°3 2°2
15 7 6 ners 0 8-0 4°8
20 a aa aa HSB eG 3-0 15
2°5 op BS Seq. LODO) 20°0 3°3 28 °6 26-2
3:0 op op -..| 33°3 39 °3 Thchy/ 33 °3 42 *4 4°6
4°0 » » 750 39 *4 18 750 39-4 5-5
Average, exclusive of | 22°9 | 12°8 32 41-4 28-9 9:9
Control |
|
142 - Mr. E. Whitley. Effect of Acid, etc.,on — [Nov. 14,
Table I1.—Actual Number of Deaths in Fresh Eggs. Eggs of 2 days old
and eggs of 10 days old with larger quantities of acid and alkali than
were employed in the experiments, the results of which are shown in
Table I. 40 eggs in each tumbler.
| |
Fresh eggs. 2 days old. 10 days old.
|
| Ist | 2nd | 3rd | 4th | Ist | 2nd} 8rd | 4th | Ist | 2nd | 8rd | 4th
day. | day. | day. | day.| day. day.| day. | day. | day. | day. | day. | day.
Controleennnercscrnt 0) iL 2 3 1 il 1 1 0 0 (0) 0
3c.c.N/10NaOH.... 0 | 2 | 11 | 25 |
4 5 55 seal) 24 || aly | HY)
5 49 5 | 0 5 | 34 | 36 0 1 1 iL 0 0 0 2
6 ; | 2) 1-29) | 36) all |
Siac’, TO Pas PO 2 |p | ae
10 $5 - IS HS eS eS eS SK ] KH LS 0) 1 2 | less
| | | | than
| 10
15 x3 55 | — — j;— J|— | — J — — |'— 0) 2 3 | less
than
| 10
PO ashes alee — {|= |] =] j— = 04) eee
30 oy) ” rae Fal ae aa aa ray aaa en ® i B 26
| — —
3 ¢.c. N/10 HCl...... @ | 1 2
7 Wy deg RR RP ana oD oi Of wy |
Be nee eee [75/8 |i som tee | 9 | 20 | 23
Strengths of 6, 8, 10, 15, 20 and 30 c.c. acid were also tried, all being fatal
in less than 24 hours.
The figures for the 2nd, 3rd and 4th days are cumulative.
1905.| the Development of Eggs of Plaice and Echinus. 143
Table ILJ.—Actual Number of Deaths among Fresh Eggs.
|
No. | 2nd]| 3rd | 4th | 5th | 6th | 7th | 8th | 9th | 10th
of eges. | day. | day.| day. | day. | day. | day. | day. | day. | day.
|
@omntrolieecers 20 1 1 1 1 il 1 2 3 5
|
| | |
1:0 cc. N/10 NaOH | 23 0 0 0 1 |
Le ae i 21 @ Weal 1 2 2
0! 5, - 20 1 4 4 5 5
AG, i 22 4 | 4 4 4 4 6 6 6 | ia
acon °.,, i Tp We aky NPGS 5 5 6 6 6 7 it
ASO 2 4 25 Shs lp atl i | ob 12 12uo| ok
|
1:0 ce. N/10 HCl...| 20 1 yD Dew 2 3 2 |
Leas f 21 Oi @ Oo ) |
BO! 4, ys 23 By | Dah e | |
BSitey |; < 21 2 3 3 3 4 5 5 Sie
350. * 24 eeOic | 6 fi 8 8 8 Shales
AO, ,; mn | 8 20 Seu iguow lp 15: 15 15 15 15 vey | || ais
| | |
The figures in each column in this and the following two tables give the
total number dead by the day in question.
Table [V.—Actual Number of Deaths among Eggs 2 Days Old.
| | |
No. of | 2nd} 3rd | 4th | 5th | 6th 7th 8th | 9th | 10th | 11th
eggs. | day. | day. | day. | day. | day. | day. | day.| day.| day. | day.
| |
| | } j
Gontroline: c.sccne ies 36 QO WON Oro oo 0 ) ft)
10cc.N/10 NaOH | 28 OW OF Cio Ole
15 i % 23 OP ON Ol O 1 OT oO
PEO, 5 a BIO 1 O | OW OA OW Oo
25 if eS 38 TU ey Pega dal ogre al alec. Wy Kalil |i a 1 1
50 awe i. 55 OU OO) NO alae seh 07 ele aS
4:0 5 56 euch ere ee Ome On lee 235) (623) 523,05 27
i —
| | j | |
10c¢c.N/10HCl ....| 60 | 1 Phe) a ae 2 2 |
Tbe be rl, Db OT lhe Pounce NHR) Salta geal |
2 Ae vaca WBS Looe Teal ae Wee! Nie
Bey, el 280 SEOs ee OnatOM eo imtGwe | tz | 177 | 19. | 21
Oe eon On 10) LOM LON 13) eis i4a 4) 14s | 14
Ue aetna 95 | 28 | 23 | 23 | 26 | 81 | 84 | 84 | 34 | 84 | 34
144 Mr. E. Whitley. Effect of Acid, etc., on [Nov. 14,
Table V.—Actual Number of Deaths among Eges 10 Days Old.
No. of | 2nd | 3rd } 4th | 5th | 6th | 7th | 8th | 9th | 10th | 11th
eggs. | day. | day. | day. | day. | day. | day. | day. | day. | day. | day.
Control ......... 245 0 0) 1 4 4, 5 6 6
1:0 c¢.c. N/10 NaOH | 165 0) 2 4 6 | 6 7
PD eee i, 105 ©}! P| ares pee sti es
20. a 110 1 Tew Peale tee rl 1
Rae i 100 le Ome tal st) i oe
BDO) g ‘4 80 CE ORAROM it ha Le Sales 2 2
AiO! - 160 Ue WS | TS SO Peet ule
10 c.c. N/10 HCl 135 Nap els | a les |
ree ‘ SOW Oke |S | 2 |e |
2°0 x) 0 155 0) 0) 0) (0) 0 iL
Bi ers cool 120 0 1 2 Silints: A |
3:0 x 0) 175 0 3 3 3) | 3 3 3 5) 6 8
Ai Oun * 110 0: ), ila |) cepa SR Ma ee Salers al 85, 5 6
Control) .scs asses eeen 98-4
+1:0 ec. N/10 NaOH ...... 96-4
15) fe Wie 99-0
2:0 Ae 2 oer 99-1
2°5 bo nt eae 99-0
30 : AA es BOCaT
4-0 Pe RNY Scitinca.5 95°6
stale OR CsCoe NI Al OMEN CR ee neces 98°5
1s SENG. avis okeeoere 96:2
& 2:0 ett TR esad ae 100-0
2S) Sub Rt gba eset dene 97-5
30 a yA ide Me Ay) 98°3
4:0 TT Se can ae 98-2
Dime thyly Ae jae en atone 39°7
Phenol-phthalein ............ 98°8
145
the Development of Eggs of Plaice and Echinus.
1905. |
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LXXVII.—B.
VOL.
150
The Mammalhan Cerebral Cortex, with Special Reference to its
Comparative Histology. I. Order Insectivora.—Preliminary
Communication.
By Grorce A. Watson, M.B., C.M. Edin,
(From the Pathological Laboratory of the London County Asylums, Claybury.)
(Communicated by Dr. F. W. Mott, F.R.S. Received July 28,—Read
December 14, 1905.)
The results and conclusions brought forward in this paper form a portion of
the outcome of an extensive investigation dealing with the cortex cerebri in
various orders of mammals. The work has special reference to the neopallium
only, and has for its prime purpose an endeavour to shed some further light
upon the functional significance of the cerebral cortical lamination.
Animals Hxamined and Methods of Study.
The brains of the animals belonging to this order examined are :—
1. The Mole (Zalpa Europea). 2. The Shrew (Sores vulgaris). 35. The
Hedgehog (Hrinaceus Huwropeus).
The cerebral cortex has been examined by means of complete series of
sections cut in almost every possible direction and stained by one or other
modification of the Nissl method.
As part of the method of study throughout the entire investigation, the
natural habits of the animals examined, and their educability, as far as facts
relating to the latter are available, have been considered when attempting to
correlate structure and function.
Macroscopic Appearances and Microscopic Furrows.
All are almost smooth highly macrosmatic brains. That of the Hedgehog
is one of the simplest mammalian brains. It presents in addition to the
rhinal fissure a short presylvian furrow; the latter is not found macros-
copically in the Mole and Shrew, but is seen on microscopic examination of
sections. By this method also, in the Mole only, two shallow more or less
longitudinal curved furrows can be traced on the dorso-lateral aspect of the
hemisphere, which appear to represent foreshadowings of the corono-lateral
and supra-sylvian sulci.
Signs of greater differentiation of the neopallium of the Mole, as compared
with the Hedgehog especially, are further exhibited on microscopic exami-
nation of the structure of the cortex. The optic nerves in the Mole and
The Mammalian Cerebral Cortex. 151
Shrew are reduced to small threads. In the Hedgehog these nerves are
considerably larger. In all three the fifth nerves are relatively very large.
The Lamination of the Neopallium.
General Remarks.—The classification of the cortical layers adopted by the
writer is that introduced by J. Shaw Bolton. The latter considers that
the human cerebral cortex is constructed upon a five-layered type—viz.,
{, Molecular; II, Pyramidal; III, Granular; IV, Inner line of Baillarger:
V, Polymorphic, Of these only three are primarily cell layers—viz., the
pyramidal, granular, and polymorphic, Layers I and IV being primarily fibre
layers, although containing nerve cells—the cells of Cajal in Layer I, and the
Betz cells (psycho-motor region) or solitary cells of Meynert (other regions)
in Layer IV. The outstanding features of this classification are: (1) The
recognition of the granular layer as separating the true pyramidal /ayer above
from the more or less pyramidal shaped cells which may be found below this
layer, for the cells of Layer IV are not “ pyramidal” cells at all, the Betz cells
in the psycho-motor area constituting “the origin of the important tract for
skilled voluntary movement,” whilst the solitary cells of Meynert in other
regions “probably possess a somewhat analogous function.” (2) The con-
sideration of the pyramidal layer as forming one layer developmentally and
functionally.
Bolton, as the result of his studies of the development of the human cerebral
cortical layers, and of their depth in the normal individual as well as in
various degrees of amentia and dementia, has come to the following conclusions
as to the functions of the three primary cell layers. The pyramidal layer
“subserves the psychic or associational functions of the cerebrum.’ The
granule layer “ probably subserves the reception or immediate transformation
of afferent impressions, whether from the sense organs or from other parts of
the cerebrum,” whilst the fifth, or polymorphic layer, “ probably subserves the
lower voluntary functions of the animal economy.”
When dealing with the mammalian cortex generally one or two further
explanations are necessary.
The term “granular” is used in a wide generic sense and as indicative of
a certain cortical layer rather than of the cell constituents of this layer,
which latter, in an adult animal, may take the form of angular, quadrilateral,
stellate or even small pyramidal-shaped cells, or a mixture of these elements.
In some regions of the cortex in certain animals these elements of the
granular layer may be scattered and comparatively few in number; yet
their recognition is of importance, for such provides the means by which
the lower limits of the true pyramidal layer may be determined. Owing
M 2
152 Mr. G. A. Watson. [July 28,
to the difficulty experienced in accurately separating the fourth and fifth
layers (which tend to intermingle) in the cortex of some animals, the writer
prefers to speak of these layers together as infra-granular. It is also
proposed for the sake of definiteness to term the true pyramidal layer (‘.c.,
Layer IT) the supra-granular layer.
Areas in the Neopallium.
The appearances of the cortex in the Mole and Shrew being very similar,
the following, concerning the cortex of the Mole, may be taken as applying
also to the Shrew excepting when the latter is specially mentioned :-—
1. Dorso-lateral Surface-—In the Mole this region presents two main and
distinct types of cortical structure, with certain areas of comparatively
undifferentiated cortex (fig. 1).
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Fic. 1.—Dorsv-lateral view of the Right Hemisphere of the Mole.
Left-hand figure. x Ayvea 1, “Motor”; x thesame, but less characteristic ; © Area 2,
General Sensory ; © the same, but less characteristic ; 5 Archipallium.
The anterior, lateral, and posterior areas of undifferentiated cortex are left blank
excepting the portion of neopallium represented as shaded, which is thinner than
the remainder.
Right-hand figure. a, vhinal fissure ; b, c, and d, probable representatives respectively
of the corono-lateral, supra sylvian, and presylvian sulci; 6, c, and d, vary much in
individual distinctness in different hemispheres. The figure is a composite one.
Area I: Motor.—This extends antero-posteriorly from a short distance
1905. | The Mammalian Cerebral Cortex. 153
behind the anterior pole to about the posterior quarter of the hemisphere,
and laterally from the dorso-mesial margin (or more or less close to this
anteriorly and overlapping this posteriorly), to about half-way between the
dorso-mesial edge and the rhinal fissure, the lateral limits varying somewhat
at different points (figs. 1 and 2).
Area IT: General Sensory.—This occupies an extensive region lateral to
Area I, but does not reach as far as the rhinal fissure, being separated from
the latter by a zone of undifferentiated cortex (fig. 1).
The features of Area I are less characteristic mesially, anteriorly and
posteriorly, and of Avea II anteriorly and posteriorly, These two fields
appear in every way to be the best developed areas of the neopallium and
to be amongst the oldest. phylogenetically. Owing to their different
histological appearances, Areas I and II in the Mole can be readily
delimited. From the relatively greater numbers and prominence of the
large cells (homologues of Betz cells) in Layer IV, it is coneluded that
Area I possesses especially motor attributes, and that on account of the
ereater development of the granular layer throughout Area II, the latter
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with fig. 2 (Mole).
Mole) and the cortex behind and below this, including archipallium on
the one hand, and the posterior part of the corpus callosum and hippo-
campus on the other. The area is not of the same shape in the Mole
as in the Hedgehog, owing to the hemisphere being moulded in a different
way in the two animals. This region, although characterised throughout
by the well-marked granular layer referred to, presents certain differences
of detail, owing to which it has been separated into three divisions
in each animal. The anterior and superior portion (Division 1) is of about
the same relative size in the Mole as in the Hedgehog. In both there are
good granular and infra-granular layers, and in the Hedgehog a shallow but
definite supra-granular layer. The middle portion (Division 2) is relatively
156 Mr. G. A. Watson. [July 28,
larger in the Mole than in the Hedgehog, but in both, comparatively to
Divisions 1 and 3, it presents unspecialised features. The inferior portion
(Division 3) is relatively not.only considerably larger in the Hedgehog than
in the Mole, but in the former it is a more histologically distinct field,
showing not only a well-formed “granular” layer (the cellular elements
in which, however, are mostly angular or small pyramidal in shape), but
a comparatively good infra-granular and a definite, though shallow, supra-
granular layer. In the Shrew this division is rather better developed than
in the Mole.
Owing to the presence throughout this region of such a deep and definite
granular layer, it is concluded that this field is sensory in function, and
regarding it the following suggestions are made :—
(a) The area is much too large to be concerned only with the cortical
distribution of the optic nerves, which are relatively so minute in these
animals, especially in the Mole.
(6) The inferior portion (Division 3) only is visual. In the Mole this
portion is a mere vestige; in the Hedgehog it is better developed and
relatively larger, having apparently extended upwards somewhat and
encroached upon the area of unspecialised granular cortex (Division 2) as
compared with the Mole.
(c) The middle and superior portions (Divisions 2 and 1) may correspond
to the large infra-calcarine area of certain relatively higher mammals (¢.¢.,
Ungulata and Carnivora), in which, owing to the greater development of the
visual faculty, the inferior portion (Division 3, visual) has, so to speak,
extended upwards, backwards, outwards, and forwards so as to overlie the
middle and superior portions, and has become the calearine region.
(d) The relatively well-developed superior and anterior portion (Division 1)
of the two specialised divisions in both Mole and Hedgehog may be concerned
with the cortical distribution of the fifth sensory nerve. On account of the
importance of the fifth sensory nerve as an avenue of information to these
mammals through snout or vibrissee touch, or both, and, in view of the large
size in them of the fifth nerve, it seems probable that the sensory portion of
this nerve should have a very special cortical representation.
The Cerebral Cortical Layers. (Neopallium.)
Although the total depth of the cortex in the best developed regions is
different in the Mole, Shrew, and Hedgehog, the relative depth of the
separate layers, supra- and infra-granular particularly, appears to be about
the same in all. The following micrometric measurements of the cortical
layers in three areas of the Mole’s cortex, which have been kindly furnished
1905. | The Mammalian Cerebral Cortex. 157
by Dr. J. S. Bolton, may be taken as fairly typical also of the relative
70 4 hours, ammonium sulphide,
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Fig. 16.—From the spinal cord of frog, illustrating a typical Frommann striation, as
revealed by the mercurous nitrate method. Hg,(NO;), =. 30 minutes,
ammonium sulphide, glycerine. x 590.
Macallum and Menten. Roy. Soc. Proc., B. vol. 77, Plate 2.
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1905.] Distribution of Chlorides in Nerve Cells and Fibres. 193
PLATE 4.
Fia. 17.—From the sciatic of frog, showing the occurrence of chlorides in the imbrica-
tions of Lanterman. x 590.
Fic. 18.—From the sciatic of frog, illustrating a distribution of chlorides in the
medullary sheath sometimes found. Hg,(NO,), e 30 minutes, ammonium
sulphide, glycerine. x 590.
Fic. 19.—From the sciatic of frog, illustrating the occurrence of chlorides in the
imbrications of Lanterman and in the axon. Hg,(NO,), = 2 hours,
ammonium sulphide, glycerine. x 70.
Fig. 20.—Portion of end of capillary tube filled with albumen. AgNO, = +15 per
cent. HNO,. The reagent has diffused in the direction of the arrow.
x 105.
Fig. 21.—Portion of another capillary tube similarly treated. x 105.
Fic. 22.—Portion of a capillary tube filled with albumen and treated as in the case of
fig. 20. x 590.
Fie. 23.—Portion of a capillary tube filled with albumen, to which sodium chloride was
added to bring the strength of the salt in solution up to 1°73 per cent.
Treated as in the case of fig. 20. x 70.
Fic. 24.—Portion of a capillary tube filled with gelatine containing 0:288 per cent. of
sodium chloride, illustrating irregularities in the distribution of the silver
precipitate. Treated as in the case of fig. 20. x 90.
Fies. 25 and 26.—Portions of capillary tubes filled with commercial gelatine and
treated as in the case of fig. 20, illustrating peculiarities in the precipitation
of the silver chloride formed. Fig. 25 x 590, fig. 26 x 105.
194
On the Possibility of Determining the Presence or Absence of
Tubercular Infection by the Exanunation of a Patient's Blood ~
and Tissue Fluids.
By A. E. Wricut, M.D., sometime Professor of Pathology, Army Medical
School, Netley ; Pathologist to St. Mary’s Hospital, London, W.; and
Staff-Surgeon 8. T. Rem, R.N.
(From the Pathological Laboratory, St Mary’s Hospital, London, W.)
(Communicated by Sir John Burdon Sanderson, Bart., F.R.S. Received
October 21,—Read November 23, 1905.)
In the present communication we propose (a) to set forth certain con-
clusions arrived at after the study of the tuberculo-opsonic power of the blood
in a very considerable number of tubercular patients; (0) to show that we
have in the measurement of the tuberculo-opsonic power of the blood and
tissue fluids a method which may be exploited in the diagnosis of tubercular
infection.
Technique Employed—tThe technique employed by us in the measurement
of the tuberculo-opsonic power of the blood was essentially that described by
one of us in conjunction with Douglas.* In each case the white corpuscles
required for the tests were derived from blood from the finger received into a
solution of 0°5 per cent. citrate of soda in 0°85 NaCl, and rewashed after
centrifugalisation in a considerable volume of 0°85 NaCl, and then again
centrifugalised. Of the “blood-cream,” obtained by skimming off the upper
layer of the corpuscular sediment, one portion was in each case mixed in a
capillary tube with one volume of serum and one volume of a suspension of
tubercle bacilli which had been centrifugalised in such a manner as to free it
from bacillary clumps. After incubation at 37° for 15 to 20 minutes films
were made on slides prepared with emery paper.t These films were, after
fixture in saturated corrosive sublimate, stained with boiling carbol-fuchsin,
decolourised with 2 per cent. sulphuric acid, and counter-stained with
methylene blue after washing in 1 in 1000 sodium carbonate. The standard
of comparison employed was obtained by mixing in each case the same
“blood cream” and tubercle suspension with “pooled normal serum.”
* ‘Roy. Soc. Proc.,’ vol. 72.
+ Wright, ‘ Lancet,’ July 9, 1904.
{ While in this research pooled serum was employed, in order to provide against any
chance variation of our bloods under the physical strain entailed by the work, it is to be
- noted that the observations of Urwick, conducted in this laboratory, and the more exten-
On Tubercular Infection in a Patient's Blood, ete. 195
This “pooled serum” was obtained by mixing equal volumes of the sera of
six to eight healthy students or laboratory workers. We have found that the
opsonic power of such a “ pooled serum” corresponds to the arithmetical mean
of the opsonic indices of its component sera.
Classification of Tubercular Cases into Strictly Localised Cases, and Cases which
are associated with Constitutional Disturbance.
Cases of tubercular infection distribute themselves in a natural manner
under two headings. Into one category would fall the patients who are the
subjects of a strictly localised infection unaccompanied by anything in the
nature of constitutional disturbance. Cases where the infection is limited to
one or more lymphatic glands; further, most cases of lupus, most cases of
tubercular abscess in the subcutaneous tissue, tubercular affections of the
joints, and, lastly, many stationary or only slowly progressing cases of
tubercular phthisis, fall into this category.
Into another category would fall patients who are suffering from more
generalised tubercular infections associated with constitutional disturbance.
This group consists in large part of cases of pyrexial pulmonary tuberculosis.
With these may be classed certain other cases of extensive or widely dis-
seminated tuberculosis.
Data with regard to the Tuberculo-opsonic Power in Cases of Strictly Localised
Tuberculosis,
The opsonic index is here low and uniformly low—in exceptional cases as
low as one-sixth of the normal. Our findings in a series of cases of strictly
localised tubercular infection are appended in tabular form below.
sive series of investigations carried out by Bulloch at the London Hospital (‘Medico-
Chirurg. Soc. Proc.,’ 1905), and Lawson and Stewart at the Banchory Sanatorium (Joc.
cit.), have conclusively shown—(a) That the tuberculo-opsonic power of the blood does
not in health range below 0'9 or above 1'1; and (6) that the bloods of A. E. W., S. R. D.,
and others which have hitherto in this laboratory furnished a standard of comparison,
are, from the point of view of their tuberculo-opsonic power, typically normal bloods.
196 Dr. A. E. Wright and Staff-Surgeon 8. T. Reid. [Oct. 21,
Table I—Showing the Tuberculo-opsonic Index in a Series of Cases of
strictly localised Tuberculosis.
Initials or, as the Tapert ;
Serial No. case may be, Nature and seat of the infection. baat doc ae
initial of patients. eee
1 J. R. Muberelelobitestisn. sr s..cccs+ sec fe eeseee- cesses 0°65
2 A. Caries, lower end of femur...............++- 0:7
3 C.8. Tubercular ulceration, dorsum of hand 0°86
4 A. B. Hs iritis. 0°51
5 B. C. _ fEllgnEGlS coooacnacdoo0aR—s590000020 0°4
6 E. M. op ulceration of legs. 13 years’
GIPENHIOSS _ ieagoseaancasacoocded ~O aly
7 H. W. By glands (neck) ............:0008 0 82
8 D. W. i “6 eM Lek id: 0°64
9 8. - ulceration of legs ............ 0-49
10 L. B. PF glands (abdominal)............ 0:13
11 iB: é AGM OV Ae eeeteaccss ies aceet sneer 0°75
12 M. H. . GYUDON) ocisosadsosdabongdboonde0 260 0°85
13 D. B. iy glands. Extirpated and re-
IOS! sooso9opdvan50o050n|A 0.85
14 M. O. #, 3) glandsteespeesceedettseeaeaciege 0°6
15 W. Psoas/abscessy didi. tlhtane ts dscesesseeeteecters 0°75
16 C. H. - sialyl dsscbanenemssuseeneies swyesectielses 0°65
17 W. Tubercular glands (neck). 18 months’
duration seeecse a. seses sete 0°47
18 C. sn. SWIRL Gem score ae: 0-85
19 U. RB. 3 glands (neck) .............0000 0-7
20 A. H. . glands. Hxtirpated and re-
BPPCALEA Je.o.scaceeneeesnene 0°54
21 Bs ” THUKO OTS — saonocnopancconcndeda 06
22 R. a abscesses and glands ......... 0°6
23 E. TDW SIS) Saqqa6sd00s00cboncoqcRan6EcdoNd00ed000600 06
24 R. Tubercle of testes and bladder ............ 0-72
25 1 Tubercular peritonitis ...................4. 0°7
26 H. 55 caries of fibula ............... 0°6
27 C. Tubercle of kidney ..............sccseeneeneee 0°88
28 W. Tubercular disease of knee ............... 0°6
29 4, % glands (neck) recurrence ... 0-66
30 8. 5 ulcer Of fOOt ...2......20..0008 0-49
31 C. 4 disease of knee .............+ 0°75
Data with regard to the Tuberculo-opsonic Power of the Blood in Cases of
Tuberculosis associated with Constitutional Disturbance.
In the cases here in question the opsonic index of the blood is continually
varying. The range of its fluctuation is from considerably under the normal
to twice or more the normal height.
Striking examples of the variation of the opsonic index in connection with
acute tubercular phthisis are furnished in the paper of our fellow-worker,
R. H. Urwick, already referred to.*
The following are instances of similar variation occurring in the subjects of
other forms of tubercular infection :—
* ‘British Medical Journal,’ July 22, 1905.
1905.] On Tubercular Infection in a Patient's Blood, ete. 197
Example 1—Child with Tubercular Caries of the Fibula, associated with
Constitutional Disturbance.
Dates of blood Tuberculo-
examinations. opsonic index.
11.9.05 1:45
14.9.05 17a
19.9.05 13
28.9.05 0:98
30.9.05 Operation, fibula scraped.
2.10.05 Tis
3.10.05 1-13
10.10.05 13
Example 2.—Adult Patient with Tubercular Caries of the Spine and
Constitutional Disturbance.
Date of blood | Tuberculo-opsonic
examination. index. Remarks.
meee Coie: os Temperature disturbance and pain associated
22.5.05........ 13 with development of abscess.
Ss OO ccwaccas’ 1°0 Temperature returns to normal in association
24.5.05..c..0e8 0°8 with spontaneous discharge of abscess.
Example 3.—Adult Patient with extensive Psoas Abscess and Generalisation
of Tubercle. Case has since terminated fatally.
Date of blood Tuberculo-opsonic
examinations. index.
8.2.05 2
9.2.05 24
11.2.05 0°6
Suggested Interpretation of the Different Findings in these two Categories of
Cases.
The explanation of the difference in the condition of the blood in these two
contrasted categories of cases is probably the following: The condition of
low opsonie power which is associated with strictly localised tuberculosis is
almost certainly a condition which has preceded and has furnished the oppor-
tunity for infection. The fact that the opsonic index continues persistently
*A rise in the opsonic power similar to this here registered has been repeatedly
observed by us in connection with the stirring up by surgical interference of tubercular
foci.
VOL. LXXVII.—B. Ip
198 Dr. A. E. Wright and Staff-Surgeon 8. T. Reid. [Oct. 21,
low after infection has supervened, while it can invariably be raised by
appropriate inoculation,* indicates that the machinery of immunisation with
which the organism is furnished is not, under the conditions which obtain in
strictly localised tubercular infections, spontaneously called into play.
The constant fluctuation in the opsonic power of the blood in cases of active
pulmonary tuberculosis and other active forms of tubercular infection
furnishes—as we can hardly doubt—evidence of a periodic conveyance of
tubercular elements into the blood ; and of a response to such stimulation on
the part of the machinery for immunisation. The low opsonic indices
registered in connection with active tuberculosis would in other words be
“negative phases” such as supervene—as one of us has shown—upon the
inoculation of all vaccines; the high opsonic indices would be “ positive
phases,” such as normally succeed upon the negative phases just mentioned ;
and the normal opsonic indices would correspond to periods of transition
between negative and positive phases, or, as the case may be, to periods
in which the blood is returning after a positive phase to the condition quo
ante.
The life of a patient with any really active form of tuberculosis would in
conformity with this view be a life of alternating negative and positive
phases: the favourable or unfavourable event of the infection being in each
case determined by the adjustment or want of adjustment of the auto-
inoculations (with respect to dosage and interspacing) with the particular
patient’s capacity for immunising response. -
Having now to a certain extent cleared the ground, we may pass on to
consider the question of the diagnosis of tubercular infection by means of the
measurement of the opsonic power of the blood.
Exploitation of the data Summarised above as an aid in the Diagnosis of
Tubercular Infection.
Consideration will make clear that the data obtained by the measurement
of the opsonic power in cases of doubtful diagnosis may, when adjudicated
upon in the light of the data obtained in connection with undoubted cases of
tuberculosis as given above, furnish material for admitting or rejecting the
diagnosis of tubercular infection. We may formulate in connection with this
matter the following propositions :—
(1) Conclusions which can be arrived at when we have at disposal the
results of a series of measurements.
(a) Where a series of measurements of the opsonic power of the blood reveals a
* Exactly the same statements hold true with regard to the staphylo-opsonic power in
localised staphylococcus infections (furunculosis, sycosis, etc.).
1905.] On Tubercular Infection in a Patient's Blood, ete. 199
persistently low opsonic power with respect to the tubercle bacillus, it may be
inferred, in the case where there is evidence of a localised bacterial infection
which suggests tuberculosis, that the infection in question is tubercular in
character.
(b) Where repeated examination reveals a persistently normal opsonic power
with respect to the tubercle bacillus, the diagnosis of tubercle may with probability
be excluded.
Illustrative case: A. B.—Case diagnosed as tubercular cystitis on the
evidence of pus in the urine, of the cystoscopic appearances and general
disturbance of health. The measurement of the tuberculo-opsonic power of
the blood yielded the following results :—
Date of blood Tuberculo-opsonic
examination. index.
2.3.05 0-98
14.4.05 0:99
28.4.05 1
18.5.05 1
19.5.05 11
2.10.05 0:97
The inference that the cystitis and disturbance of health was not of
tubercular origin was confirmed (a) by the fact that an extensive series of
bacteriological examinations prolonged over many months revealed in every
case the presence of proteus in large numbers, while the tubercle bacillus was
never found, even when examined for by the inoscopic method of Jousset;
(b) by the fact that the patient’s blood possessed, anterior to treatment with
regard to the proteus, an agglutinating power which was three times higher
than the normal; and (c) by the fact that very striking amelioration of the
cystitis, and a complete return to health has been obtained as the result of
the inoculation of a proteus vaccine.
(c) Where there is revealed by a series of blood examinations a constantly
Jluctuating opsonic index the presence of active tuberculosis may be inferred.
C. D—A case of severe chronic urticaria of unknown etiology. The
measurement of the tuberculo-opsonic power of the patient’s blood yielded
the following results :—
Date of blood §Tuberculo-opsonic
examination. index.
20.5.05 13
26.8.05 13
16.6.05 0°86
20.6.05 1:27
200 Dr, A. E. Wright and Staff-Surgeon S. T. Reid. [Oct. 21,
The inference drawn from these data that the patient was suffering from
some active form of tuberculosis was confirmed (a) by the discovery by an
independent observer of a lesion in the apex of one lung; (0) by the subsequent
development of an abscess of an obviously tubercular character; and (c) by
the marked improvement in health which has followed upon inoculation with
tubercle vaccine.
(2) Conclusions which may be arrived at where we have at disposal the
result of an isolated blood examination.
(a) Where an isolated blood examination reveals that the tuberculo-opsonic power
of the blood is low, we may—according as we have evidence of a localised bacterial
infection or of constitutional disturbance—infer with probability that we are
dealing with tuberculosis—in the former case with a localised tubercular infection,
in the latter with an active systemic infection.
(b) Where an isolated blood examination reveals that the tuberculo-opsonic
power of the blood is hiyh, we may infer that we have to deal with a systemic
tuberculous infection which is active, or has recently been active.
(c) Where the tuberculo-opsonie power is found normal, or nearly normal,
while there are symptoms which suggest tuberculosis, we are not warranted,
apart from the further test described below, in arriving at a positwe or a
negative diagnosis.
Discrimination of Tubercular Blood from Normal Blood by the ad of the
Phagocytic Test Conducted with Serum which has been sulyected to a
Temperature of 60° C.
The further criterion to which reference was made in the preceding
paragraph is the following :—
When a serum is found to retain in any considerable measure, after it has
been heated to 60° for 10 minutes, its power of inciting phagocytosis we may
conclude that “ incitor elements”* have been elaborated in the organism either m
response to auto-inoculations occurring spontaneously in the course of tubercular
infection, or, as the case may be, under the artificial stumulus supplied by the
inoculation of tubercle vaccine.
A typical selection from the very extensive body of observations which
furnishes the basis of the above statement is presented in Tables II
and III.
It will be seen from these tables that in practically every case where a
reaction to tubercular infection may be assumed to have taken place,
* The term “incitor elements” (Latin, incito, I urge forward, I hasten, I bring into
rapid movement) is here employed in lieu of a more specific term, in order not to prejudge
the mode of action of the element in the heated serum which promotes phagocytosis. The
nature of the incitor element is considered in the next following communication.
1905.] On Tubercular Infection in a Patient's Blood, ete. 201
Table I1.—Showing that the Normal Serum, after it has been exposed to a
Temperature of 60° C. for 10 minutes, no longer incites Phagocytosis.*
Unheated serum. Heated serum.
Serial i
number| Derivation of the | Phagocytic count. Phagocytic count.
of the eae (Number of Tuberculo- (viele oe Tuberculo-
observa- bacteria ingested . bacteria ingested :
tion divided b papi divided b Weise
: y index. y index.
number of leuco- number of leuco-
cytes examined.) cytes examined.)
1 Healthy man ......... (104/40) = 2°6 | Takenas1 Caio} = 0°32 0°125
PA allie? Ua) Sel SanPep rane (96/40) = 2°4 1 8/40) = 0°2 0:08
3 Pooled serum of six (247/86) = 6°8 i 1.| (30/50) = 0°6 0:09
healthy men
4 | Healthy boy............ (250/39) = 6-4 Gt ae = 0 a 0:06
AN a ae a (214/30) — 7-0 1 | (19/40) = 0-4 0-06
6 Pooled serum of eight| (60/50) = 1:2 i 1} (2/20) = 0°71 0-08
normal men
7 Healthy man ......... (55/40) = 1°4 55 1} (0/40) =0-0 0:00
8 Pooled serum of six | (182/80) = 4°4' of 1} (8/30) = 071 0-1
healthy men
evidence of that reaction can be obtained by conducting the phagocytic test
with serum which has been heated to 60° C. for 10 minutes.
The observations numbered 15 and 16 respectively have, it may be noted,
been introduced into the table with the special design of showing the very
simple nature of the investigation which is required for the diagnosis of
tubercle in the case where that infection has called forth a reaction of
immunisation.
The following observations, which we owe to our fellow-worker
Dr. G. W. Ross, bring out in an instructive manner the trustworthiness
of the phagocytic test with heated serum as applied in this its simplest
form :—
Case 1.—Girl, et. six years, Tentatively Diagnosed Pulmonary Phthisis.
Phagocytosis obtained with the serum, heated for 10 minutes to 60° C. and
employed in a phagocytic mixture containing over 1 per cent. NaCl.
The verdict of tubercular infection of the lung which was based on this
was confirmed on post-mortem examination.
* In order to avoid the fallacies associated with spontaneous phagocytosis (wde the next
following communication) the observations which are recorded in this and in the subsequent
table were in each case made by mixing the volume of the serum with one volume of
corpuscles, washed in 0°85-per-cent. NaCl solution and one volume of tubercle bacilli
suspended in a 1*5-per-cent. NaCl solution. In this manner a salt content of over 1-per-.
cent. NaCl was achieved in the phagocytic mixture.
202
Dr. A. E. Wright and Staff-Surgeon 8. T. Reid. [Oct. 21,
Table III.—Showing that an element which incites Phagocytosis is contained
in the heated serum of patients who are the subjects of an active
systemic tubercular infection, or who have been subjected to inocu-
lations of a tubercle vaccine.
The sera, like those which are in
question in Table II, were in each case heated to 60° C. for 10 minutes.
Serial
number
of obser-
vation.
Ne) {e0) “TO Or iB Co bo H
ra
f=)
a
e
12
13
14
15
16
Nature of infection.
Tubercular caries of
hip
Tubercular phthisis ...
oP)
5; pe
Tubercular peritonitis
» OO
Phthisis and tuber-
cular glands
Tubercular caries of
hip
Tubercular abscess of
kidney :
Lupus under treat-
ment by inoculation
of tubercle vaccine
Tubercular ulcer of
leg under treatment
by inoculation of
tubercle vaccine
Tubercle of kidney
under treatment by
inoculation of
tubercle vaccine
Tubercular glands
and abscess under
treatment by inocu-
lation of tubercle
vaccine
Tubercular cystitis
under treatment by
inoculation of
tubercle vaccine
IPhthisisiesesecase see:
Unheated serum.
Tuberculo-
opsonic
: index
Phagocytic | (determined
(Number of By bee
bacteria Haaeeetic
ingested divided | P nae!
by number of : if
leucocytes bes a
examined.) Se
with pooled
blood of
healthy men).
= 15
(125/20) =6:2) 14
.| (152/80) = 5-0; 12
(98/30) = 3-2 10
(144/30) = 4:8 14
| (142/30) = 4°7 1°4
(113/40) = 2°8 et
(110/30) = 3°6 1-0
_ Wy
(34/10) =3-4| 0-7
(249/40) = 6-2 1
i)
(68/40) = 1-7 15
(59/40) = 1°5 1°4
(97/50) = 2-0 =
Heated serum.
Tuberculo-
opsonic
index
Phagocytic | (determined
count. by compari-
(Number of son of
bacteria phagocytic
ingested divided count
by number of with that
leucocytes obtained with
examined.) unheated
pooled
blood of
normal men).
= 0-4
(113/30) = 3-7 0°8
(96/30) = 3-2 0-72
20/65) =0-°3| O-1
(103/30) = 3-4 10
(16/50) = 0°3 0-09
(79/50) =1°6| 0-6
(85/30) =2°8) 08
(26/30) =0'8| 0-4
(49/30) = 0:33
(149/40) = 3:7 0-7
|
(77/40) = 1°9 1cY/
(36/40) =0-9| 0°8
(43/30) = 1-4 =
|
(26/80) = —
(9/5) = =
1905.| On Tubercular Infection in a Patient's Blood, etc. 208
Case 2.—Man, et. 41, Tentative Diagnosis, Pleuwrisy due to Malignant
Disease, or Tubercular Pleurisy.
No phagocytosis obtained with the serum, heated for 10 minutes to 60° C.
and employed in a phagocytic mixture containing over 1 per cent. NaCl.
The verdict of pleurisy due to malignant disease, which was based on this,
was confirmed on post-mortem examination.
Case 3.—Case Tentutiely Diagnosed Miliary Tuberculosis or Malignant
Endocarditis.
No phagocytosis obtained with the serum, heated for 10 minutes to 60° C.
and employed in a phagocytic mixture containing over 1 per cent. NaCl.
The verdict of malignant endocarditis which was based on this was
confirmed on post-mortem examination.
Observation 4.—Case Diagnosed Miliary Tuberculosis.
No phagocytosis obtained with the serum, heated for 10 minutes to 60° C.
and employed in a phagocytic mixture containing over 1 per cent. of NaCl.
The post-mortem examination revealed a complete absence of tubercular
lesions and a healing typhoid ulcer* in the ileum.
On two other Methods by which a Diagnosis of Tubercular Infection can be
arrwed at or Hxcluded.
In addition to the methods which have been already considered, there are
two further methods which can be exploited in connection with the
diagnosis of tubercular infection. The first of these is applicable where
we desire to supplement the often ambiguous data furnished by the clinical
symptoms in the case of inoculations of tuberculin undertaken for diagnostic
purposes. The second is applicable where we can obtain, in addition to the
patient’s blood, also lymph, or, as the case may be, pus from the seat of
infection.
Diagnosis of Tubercular Infection by the Aid of Measurements of the Opsonic
Power carried out in Connection with the Inoculation of Tuberculin for
Diagnostic Purposes.
Already, three years ago,f in connection with a paper on staphylococcus
inoculations as applied to the treatment of acne, furunculosis, and sycosis,
attention was directed by one of us to the close analogy between the
tubercular reaction of Koch and the local inflammation and _ general
constitutional disturbance which may supervene when a patient whose
* A negative Durham-Gruber reaction had been obtained in this case.
+ ‘Lancet,’ March 29, 1902.
204 Dr. A. E. Wright and Staff-Surgeon S. T. Reid. [Oct. 21,
tissues are extensively invaded by the staphylococcus is inoculated with the
corresponding vaccine in such a manner as to develop a pronounced
negative phase.
The association of a negative phase with a reaction similar to that
conveniently spoken of as the tuberculin reaction, suggested to us the
propriety of enquiring whether the true tuberculin reaction, as seen after
the injection of Koch’s old tuberculin into a tubercular patient, was also
associated with a negative phase.
The opportunities for investigating the question which have presented
themselves have not yet been sufficiently numerous to allow of our formulat-
ing a final answer to this question. The observations which are set forth
below seem to us to suggest that the development of a negative phase, with a
dose of tuberculin smaller than that which would produce this result in a
healthy patient, may prove to be an index of tubercular infection. Such a
conclusion would be in harmony with our experience in connection with the
therapeutic inoculation of tubercle vaccine (new tuberculin). We find in this
connection that the negative phase supervenes upon very much smaller doses
and persists much longer in the case where the patient is the subject of
extensive infection than in the contrary case.
Observation 1.—Case diagnosed, Tubercular choroiditis.
Date. | Tuberculo-opsonic index. Clinical data.
26.4.0B...s..00c0. | 0:9
5 milligrammes old tuberculin inoculated.
0-29 Some constitutional re-
0°95 action, ¢. 100° F.
Observation 2.—Case diagnosed, Lupus erythematosus.
Date. | Tuberculo-opsonic index. Clinical data.
TOO, ee | 0-73
13.1.05............ 0°85 No rise of temperature
OD eeeer cece 1°6 or constitutional or
A2GERODweaseasences 0°5 local reaction.
1905.] On Tubercular Infection in a Patient's Blood, etc. 205
Observation 3.—Case diagnosed, Lupus erythematosus.
Date. Tuberculo-opsonic index. Clinical data.
O34: Operarsgess ses 0 “66
Tnoculation of 5 milligrammes of old tuberculin. |
|
LALO: recksssnes 0-7 | Quite insignificant con-
NDA Ob secccceses 1°2 | stitutional disturbance.
TAA OD... cwacecices 0°85
Observation 4.—Case diagnosed as Lupus vulgaris.
Date. Tuberculo-opsonic index. Clinical data.
Inoculation of 5 milligrammes of old tuberculin.
PAO bi oenccncases: iL ct Quite insignificant con-
12.4.05............ 1:0 | stitutional reaction.
14.4.05.........00. 1°0 |
Observation 5.—Lupus, patient had been treated for many months by
therapeutic inoculations of tubercle vaccine.
Date. Tuberculo-opsonic index. Clinical data.
PAP O55 kad, | 1°4
Inoculation of 30 milligrammes of tuberculin.
QDs ODT eee rece | 0°34 Severe constitutional and
ZOM OSS cercccsasel 21 local reaction, ¢. of
PELOo ee 17 103° F.
Diagnosis of Tubercular Infection by the Comparison of the Opsonic Power aj '
the Patient's Blood with the Tuberculo-opsonic Power of the Fluids
Derived from the Focus of Infection.
Attention has already been drawn by one of us, both in a research
undertaken in conjunction with Lamb* and in a research undertaken in
conjunction with Douglas,f to the fact that we have in the actual focus of
* ‘Lancet,’ December 23, 1899.
+ ‘Roy. Soc. Proc.,’ vol. 74, p. 157.
206 Dr. A. E. Wright and Staff-Surgeon S. T. Reid. [Oct. 21,
infection a lowered “bacteriotropic pressure” which accounts for the
cultivation of the pathogenetic microbe in the interior of an organism which
has at disposal in the circulating blood a considerable reserve of anti-
bacterial substances. We propose here in conclusion to furnish further
illustration of the general law as enunciated above, culling our examples
not alone from the observations we have made in connection with tubercular
infection, but also from observations made in connection with other bacterial
infections.
Observation 1.—Case of abscess in the neighbourhood of the appendix,
Blood from the patient’s finger and pus obtained from the abscess at the
operation were examined, with a view to determining the nature of the
infection.
Phagocytic counts.
With a suspension of | With a suspension of
tubercle bacilli. staphylococci.
PSEISUER Socnoaccasssa0oGnod8ace. 2°3 45
Fluid obtained from the 0-1 1-9
pus by centrifugalisation
The fact that the tuberculo-opsonie power of the patient’s blood was
here 23 times as great as that of the fluid obtained from the pus was taken
as evidence that tuberculo-opsonic substances had been used up in the pus
and that the patient was suffering from a tubercular infection. It was
inferred on similar grounds that he was also infected by staphylococcus.
Observation 2.—Case of osteo-myelitis of the femur. Blood from the patient’s
finger and pus obtained from the abscess at the operation were examined,
with a view to determining the nature of the infection.
Tuberculo-opsonic Staphylo-opsonic
index. index.
S{zPAUEIL. “Ggaqaqneqoo0090900000000 1:0 2°5
Fluid obtained from the 1-1 0-9
pus by centrifugalisation
The fact that the opsonic index of the patient’s circulating blood was here
normal to tubercle, while it was two and a-half times greater than normal
1905.] On Tubercular Infection in a Patient's Blood, etc. 207
with respect to the staphylococcus, was taken as evidence that the patient
was not infected with tubercle, and that he was infected by staphylococcus,
and had responded to that infection by a production of immunising
substances.
The fact that the tuberculo-opsonic index of the fluids obtained from the
pus was the same as that of the blood, while the staphylo-opsonic power
was only two-fifths of that of the circulating blood, was taken as of con-
firmatory evidence of the conclusion already arrived at. The fact that a
copious culture of staphylococcus aureus was obtained from the pus, planted
out with aseptic precautions at the operation, further confirmed the diagnosis.
Observation 3.—Case of psoas abscess. Blood from the patient’s finger and
pus from the abscess were examined.
Phagocytic counts.
With a suspension of | With a suspension of
tubercle bacilli. staphylococci.
SEVIER, socgrasdscousondcupenede 2-4, 5°0
Fluid obtained from the 1°23 1:2
pus by centrifugalisation
The fact that the fluid obtained from the pus was impoverished in both
tuberculo- and staphylo-opsonic substances as compared with the blood was
taken as evidence of a combined infection by tubercle bacilli and staphylo-
cocci. This inference was confirmed by the fact that the opsonic power of
the blood with respect to both the micro-organisms here in question was
undergoing perpetual fluctuations.* The inference so far as it related to the
staphylococcus was further confirmed by the fact that cultures of the micro-
organism were obtained from the pus.
Observation 4.—Case of ascites with grave constitutional disturbance in
a man of 30. Blood from the finger and ascitic fluid were examined on two
occasions.
First Occasion.
Tuberculo-opsonic index.
DOWUMG ce Feces ove debited 1:05
ANSOMBOTINCL asec ancpese 0:99
We reported upon this that the patient was not suffering from tubercular
peritonitis.
* For the variations registered in connection with the tuberculo-opsonic power, vide
supra, p. 196 of this paper, where Example 3 refers to the patient here in question.
208 Dr. A. E. Wright and Staff-Surgeon 8. T. Reid. [Oct. 21,
The clinical symptoms, the age of the patient, and the appearances as seen
at the operation appearing in contradiction with this verdict, and the ascites
having reappeared, a second operation was performed, and a further sample
of ascitic fluid was obtained for examination. At the same time the clinical
appearances were again observed, with the result that there was now some
wavering as to whether the original diagnosis could be upheld. The result
of the phagocytic examination of the ascitic fluid, and of a sample of blood
from the fingers were now as under :—
Tuberculo-opsonic index.
Seralt Giescecsaeme sveees 1
In view of this result the verdict previously given was sustained.
A post-mortem examination, which followed in the course of a few weeks,
again threw doubt on the verdict, the naked-eye appearances being entirely
consistent with the theory of miliary tuberculosis affecting the peritoneum
and serous covering of the intestines. Microscopic examination of the
sections made through the miliary nodules revealed, however, a typical
picture of miliary carcinoma. No primary carcinomatous focus had been
discovered, though it was sought for on post-mortem examination.
Observation 5.—Case of pleural effusion. Blood from the finger and fluid
obtained by paracentesis of chest were examined :—
Tuberculo-opsonic index.
NOPE psp -eeeeapeenesene 0:92
Pleural (fluid) -s325-6-.2: 10
This was taken as evidence of the absence of tubercular infection.
Observation 6.—Case diagnosed as peritoneal tubercular peritonitis com-
plicated with pleurisy. Blood from the finger was examined on two
occasions. On the second occasion, which was 48 hours after the first
examination, peritoneal and pleural fluid were also examined.
The results obtained by the phagocytic examination undertaken on this
second occasion were as follows :—
Tuberculo-opsonic index.
Seruin jie yeasts 0:7
Peritoneal fluid ......... 0:28
Rleuralstiurdeseee eee il
The results of the comparison of the peritoneal fluid with the serum
obtained from the blood withdrawn from the finger were taken as evidence
of tubercular infection of the peritoneum. Confirmatory evidence of
tubercular infection was furnished further by the low tuberculo-opsonic
1905.| On Tubercular Infection in a Patient's Blood, etc. 209
power of the blood, and by the observed fluctuation in this index. When
it was measured two days previously, this index had worked out as 1°4.
The fact that the opsonic power of the pleural fluid worked out as higher
than the opsonic power of the serum was taken as evidence that the
pleural effusion had occurred at a period when the opsonic power of the
blood was 1 or above 1.
The diagnosis of tubercular infection of the peritoneum and pleura (and
underlying lung) was confirmed at the post-mortem examination.
Observation '7.—Case of long-continued suppuration of the antrum associated
with the presence in the pus of the pneumococcus and the Bacillus fusiformis
and Spirillum bucee of Vincent. The patient had been treated by
therapeutic inoculations of a pneumococcus vaccine. The patient’s serum and
the antral pus were examined with a view to determining whether the
pneumococcus played any active part in connection with the continuance of
the suppuration :-—
Pneumo-opsonic index,
SOMITE ete ida. a MAIR we'side nM eM Maes bot debininc tceei we 4:3
Fluid obtained from pus by centrifugalisation ...... 0°3
The results were taken as evidence (a) that the pneumococcus played
an active réle in connection with the suppuration, and (0) that the
protective substances which had been generated in the blood under the
influence of inoculation did not come satisfactorily into application upon the
micro-organisms in the antrum.
Observation 8.—Case of whitlow associated with the formation of a blister
under the nail. Serum derived from blood from a sound finger and blister
fluid were examined.
Staphylo-opsonic index.
SEIT Bbaboecodeanse see A 0:8
IBiStera shld weemeeese eee 0:3
The blister fluid yielded a pure culture of staphylococcus.
Observation 9.—Rabbit in the early stages of anthrax infection —Blood
obtained from the ear and lymph from the seat of inoculation were examined.
Anthraco-opsonic index.*
SOLU ya chest eat ans duetue Tey
MyM soos. showeaesecseper 0°62
* Tested with a suspension of anthrax spores and compared with the serum of a normal
rabbit tested in the same mannet.
It may be noted that all the difficulties and inaccuracies which are associated with
the employment of ordinary anthrax cultures in phagocytic experiments can be
satisfactorily evaded by the employment of suspensions of anthrax spores. These, when
stained with carbol fuchsin and decolourised by 0°25 per cent. sulphuric acid, represent
absolutely ideal elements for enumeration.
210 On Tubercular Infection in a Patient’s Blood, etc.
APPENDIX.
A further Series of Observations showing that Phagocytosis is obtained with
the Heated Serum of Patients who are the subjects of a Systeme as
distinguished from a strictly Localised Tubercular Infection, or who,
being the subjects of a strictly Localised Tubercular Infection, have been
subjected to Inoculations with Tubercle Vaccine. The serum was in each
case heated to 60° C. for 10 minutes.
Table supplementary to Table I1—Showing that the Normal Serum, after it
has been exposed to a Temperature of 60° C. for 10 minutes, no longer
incites Phagocytosis.
Unheated serum. Heated serum.
: Opsonie
ae index
Phagocytic (determined Phagocytic Gee ee
. count. by compari- count. y basse
Pali - (Number of son of (Number of phaede sie
number i F bacteria hagocytic bacteria
of obser- Natnre of infection, ingested divided 5 cuit ingested divided are t
vation. by number of with that by number of be : 4
leucocytes obtained leucocytes PA a
examined.) with pooled examined.) we he q
blood of bl i ne
healthy men). ea 2
normal men).
1 Fibroid phthisis, tu- | (100/30) = 3:3 1-0 (142/37) = 4:0 1:2
bercle bacilli in
sputum
2 | Early phthisis, tu-| (132/80) =4'4| 1°83 (122/47) = 2°6 0°77
berele bacilli in
sputum
3 Acute phthisis, tu- | (180/80) = 4:3 1°3 (96/40) = 2°4 0°74,
bercle bacilli in
sputum
4 | Acute phthisis ......... (127/40) = 3-2 1-0 (45/34) =1°3| 0-4
5 | Fibroid phthisis (?) ...| (182/30) = 6 0 1°8 (51/48) = 1-2 0:3
6 Phthisis, tubercle | (117/30) = 3°9 1:1 (65/30) = 2-2 0-62
bacilli in sputum
7 Mitral stenosis ......... (106/30) = 3°5 1°0 (19/31) = 0°6 0:17
8 Early phthisis ......... (161/30) = 5°4 1°6 (54/27) = 2°0 0°6
Q | Phthisis .......c..cc0000 (257/40) = 6°4| 1:3 51/40) =1°3| 0:27
10 Lupus under treat- | (131/36) = 3:3 16 (74/40) = 1°8 0°'8
ment by inoculation
of tubercle vaccine
11 Lupus under treat-| (73/380) = 2°4 1:2 (81/80) = 1-0 0°5
ment by inoculation
of tubercle vaccine
12 Tubercular ulcer of | (63/30) = 2:1 1:2 (60/30) = 2:0 11
leg under treatment
by inoculation of
tubercle vaccine
The first eight of the observations here in question were made upon bloods collected for us in
the Victoria Park Hospital by our fellow worker, Dr. G. W. Ross. The clinical diagnosis which
had been arrived at was not made known to us till afterwards, when the particulars set forth in
Column 2 were filled in by Dr. Ross.
On Spontaneous and other Phagocytosis. 211
In contrast with the observations incorporated in Table II in the body of
the paper these observations were conducted in phagocytic mixtures
containing 0°85 per cent. instead of 1:1 per cent. of NaCl. It is shown
in the next following communication that spontaneous phagocytosis is
absolutely abolished only in the case when the salt content of the phagocytic
mixture exceeds 1 per cent.
The source of fallacy to which attention is here called falls, no doubt, for
all practical purposes, entirely out of account.
On Spontaneous Phagocytosis, and on the Phagocytosis which is
Obtained with the Heated Serum of Patients who have
Responded to Tubercular Infection, or, as the case may be, to
the Inoculation of a Tubercle Vaccine.
By A. E. Wricut, M.D., sometime Professor of Pathology, Army Medical
School, Netley, Pathologist to St. Mary’s Hospital, London, W., and
Staff-Surgeon 8. T, Rem, R.N.
(From the Pathological Laboratory of St. Mary’s Hospital, London, W.).
(Communicated by Sir John Burdon-Sanderson, Bart., F.R.S. Received
October 21,—Read November 23, 1905.)
It has been indicated in the foregoing paper than an incitor element* is to
be found in the blood of those who have made an immunising response to
tubercular infection, or, as the case may be, to an inoculation of a tubercle
vaccine. This fact does not stand by itself.
Recital of Previous Observations on the same Subject.
The observations of Metehnikoff, following in sequence upon the classical
researches of R. Pfeiffer on the intraperitoneal destruction of bacteria by the
aid of immune sera, first drew attention to the fact that very active phagocy-
tosis comes under observation when bacterial cultures, or as the case may be
Spermatozoa, are introduced into the peritoneal cavity of normal animals in
association with heated serum derived from immunised animals.
* The term “incitor-element” (Latin—zncito: I hasten, I urge forward, I bring into
rapid movement) is here employed to denote the element in the heated serum which
promotes phagocytosis. By employing this term, pending the elucidation of the nature
and mode of action of the element in question, we secure the advantage of leaving these
issues unprejudged.
212 Dr. A. E. Wright and Staff-Surgeon 8. T. Reid. [Oct. 21,
Savtschenko* obtained in experiments conducted in vitro with the heated
sera of animals which had been subjected to injections of red blood corpuscles,
phagocytosis of these formed elements.
Neufeld and Rempau,t working with heated sera derived from animals hel
had been immunised against streptococcus and pneumococcus, and conducting
their experiments in vitro, have described these immune sera as possessing a
power of inciting phagocytosis. This power was, be it remarked, not
numerically measured.
Leishman,t employing the numerical method for the measurement of
phagocytosis which was devised by him with the modifications introduced
by one of us in conjunction with Douglas, ascertained that the sera derived
from Malta fever convalescents, or as the case may be from men who had
undergone anti-typhoid inoculation, retain, after heating, elements which
promote phagocytosis.
Dean, working with the same methods, without however conforming to the
easily realised conditions§ which are essential to the accuracy of the
enumeration, has described incitor elements in the heated serum derived
from animals which had been immunised against staphylococcus.
Lastly, Douglas, employing again the same methods, has obtained evidence of
the presence of an incitor element in the heated serum derived from himself
and others after inoculation with a sterilised culture of the plague bacillus.
Views of the Observers above mentioned on the Nature of the Incitor Element
contained in the Heated Serum.
Influenced by the theoretical conception that the increased resistance to
bacterial invasion which is obtained by bacterial inoculation is in every case
referable to a modification of the phagocytes,|| Metchnikoff originally spoke
of the incitor element as a stemulin.
* © Annales de l'Institut Pasteur,’ 1902.
+ Neufeld and Rimpau’s paper was published in the ‘ Deutsche Med. Wochenschrift’ in
September, 1904, 12 months after the first description of the opsonins in these
‘ Proceedings.’
t ‘Path. Soc. Trans.,’ 1905, vol. 56.
§ “I should not feel disposed,” remarks this author (‘ Roy. Soc. Proc.,’ Series B, vol. 76,
p. 511), “to place quite the same reliance as Wright and Douglas on the numerical
accuracy of the results which can be derived from their method. Where the leucocytes
are very full, z.¢., where the counts are high—it is impossible to differentiate results by
the method of enumeration.” In spite of the perfectly self-evident experimental limita-
tion of our method, which Dean here recognises, this worker employs in practically all
his published experiments bacterial suspensions which give him an average phagocytic
count often of 50 and more bacteria in the leucocyte. Such a count is altogether
incompatible with accurate quantitative work.
|| The correctness of the view that artificial immunity depends upon a modification of
1905. | On Spontaneous and other Phagocytosis. 213
This appellation may, we think, be characterised as unfortunate, jirst,
because the mode of action of the incitor element was prejudged ; secondly,
because the appellation suggests (in contravention to everything which has
come to light with respect to immunisation) that there are elaborated in the
animal organism in response to inoculations, not vaccinotropic elements
(elements which have a chemical affinity for the vaccine) but lewcocytropic
elements (substances which have a chemical action on leucocytes).
At a later date the terms “sensitiser ” and “fixing substance” (Ja substance
sensibilitrice and le fixateur) were applied by Metchnikoff to the incitor
element. This nomenclature is, it seems to us, almost equally infelicitous—
infelicitous because it imposes upon the mind the following ideas :—(q) that
the phenomena of phagocytosis are analogous to those of hemolysis ;
(>) that the incitor substance, like the “ amboceptor” of Ehrlich, exerts its
specific effect only in the case where it is reinforced by a complement; and
(c) that the mechanical movements of the phagocyte in the ingestion of
particulate matter are analogous to the chemical action of the complement in
the case where red blood corpuscles are dissolved by a hemolytic serum.
With the exception of Leishman,* who, with a view to conforming to the
original nomenclature of Metchnikoff, and also because his own experi-
ments incline him to adopt the same point of view, speaks of the incitor
substances as stimulins, all the other observerst take the view that the
the leucocytes was first inquired into by Denys and Leclef (‘ La Cellule,’ 1895, vol. 11), in
connection with their experiments conducted on rabbits with streptococcus. The doubt
with regard to the correctness of Metchnikoff’s view which found expression in the
paper of these authors was further justified by the experiments of Mennes (‘ Zeitsch. f-
Hygiene, 1897, vol. 25), conducted with the blood of animals immunised against the
pneumococcus. Finally, the incorrectness of the view that immunisation depends on a
modification of the leucocytes was for the first time unambiguously established by one of
us working in conjunction with Douglas (‘ Roy. Soc. Proc.,’ vol. 72, p. 369, and vol. 73
p.129). Our results were afterwards confirmed by Bulloch (‘ Roy. Soc. Proc.,’ vol. 75).
* Loe. cit. and ‘ Journ. of Hygiene,’ 1895.
+ It may be remarked in this connection that Neufeld and Rimpau, while satisfied that
the incitor substances in the serum exert an opsonic action on the bacteria, suggest that
the term opsonins should be here rejected and that the substances here in question
should be called bacteriotropins. Pending the discussion of the questions of the mode
of action of the incitor elements in the heated serum, and of their identity or non-
identity with the opsonins found in normal blood, it will suffice here to remark with
respect to the proposed nomenclature of Neufeld the following :—
(a) The term bacteriotropins (since it connotes nothing more than the property of
entering into chemical combination with bacteria) is more appropriate as a generic term
for the whole class of substances which combine chemically with bacteria, than as a
specific designation for the substances which prepare the bacteria for phagocytosis.
(6) All considerations of the comparative merits of Neufeld’s terminology and my
terminology apart—there must, I apprehend, remain to me as the author of the term
VOL. LXXVII.—B. Q
214 Dr. A. E. Wright and Staff-Surgeon S. T. Reid. [Oct. 21,
incitor element in the immune serum exerts an opsonic action upon the
bacteria, preparing them for phagocytosis.
Sourees of Fallacy which must be Eliminated before the Question as to the
Nature of the Incitor Element in the Heated Serum can be Properly
Investigated.
Before an inquiry into the nature of the incitor constituent of heated
“immune serum” can be properly taken in hand, the sources of fallacy
which are incident to such an inquiry must be realised. A first source of
fallacy is associated with the occurrence of spontaneous phagocytosis. 3000/1.
Fic. 4.—Diagram of a somatic amphiaster, in which longitudinally split V-shaped
chromosomes, with limbs of unequal length, are apparently arranged parallel to the
spindle axis. Adjacent chromosomes, with their longer limbs on opposite sides of
the equator, if regarded as together forming one chromosome, would convert such a
mitosis into a heterotype with half the somatic number of chromosomes arranged
longitudinally on the spindle, e.g., figs. 3, 5, 6, 7, 8, 9, and 10.
Fies. 5 and 6.—Apparent heterotypical mitosis. Fig. 5, replica of fig. 4, Royal Society
paper, and of fig. 26 in First Scientific Report, 1904. Transplanted carcinoma of
* ©Centralb. f. Path. u. path. Anat.,’ vol. 18, 1902.
+ Loe. cit.
232 On the Occurrence of Heterotypical Mitoses in Cancer.
mouse. Chromosomes arranged longitudinally on the spindle. The mitosis is
contained in two consecutive sections. 3000/1.
Fies. 7 and 8.—Same sections as figs. 5 and 6. Result of analysis after restaining.
Fic.
Fie.
Longitudinally split chromosomes with unequal limbs projecting above and helow
the equatorial plane. X 3000/1 of diagram, fig. 4.
9.—Apparent heterotypical mitosis. Transplanted carcinoma of mouse. Loop and
figure-of-8 chromosomes arranged longitudinally on the spindle. > 3000/1.
10.—Analysis of the same preparation as fig. 9, showing the slight differences in
interpretation sufficient to make this mitosis conform to the somatic type. The loop
chromosome in the middle of the equatorial plate consists of two distinct V-shaped
.chromosomes with unequal limbs projecting above and below the equator. The
Fic.
Fic.
PIG.
Fic.
attraction fibres are attached to the apices, and not to the ends of the long limbs as
would be the case in a true heterotype. X 3000/1.
3. 11.—Diagram of a somatic metaphase in which the limbs of the chromosomes are of
unequal length. The longer limbs still cohere after separation of the apices and
shorter limbs. The barrel-shaped figure thus produced resembles a heterotype,
especially when the compressed form of the cytoplasm crowds the chromosomes
together.
12.—Shows the detailed analysis of the mitosis at the upper part of fig. 20, Plate 7,
Second Scientific Renort, 1905. It illustrates the mode of separation of daughter
chromosomes with unequal limbs, as represented diagrammatically in fig. 11.
Transplanted carcinoma of mouse. X 3000/1.
13.—Microphotograph (untouched) of “monaster” mitosis from squamous-celled
carcinoma of the tongue (man). Shows ring and U-shaped chromosomes. 1000/1.
14.—Analysis of same section as fig. 13. Partial separation of the daughter
chromosomes accounts for the presence of ring and U-shaped chromosomes. No
centrosomes or achromatic figure visible. X 3000/1.
15.—Remainder of same cell in next section. Shows large number of chromosomes
of ring and U-shape, along with others in which the widely separated daughter-rods
are parallel to each other. x 3000/1.
Bashford and Murray. Roy. Soc. Proc., B. vol. 77, Plate 5.
7 Bashford and Murray. Roy. Soc. Proc., B. vol. 77, Plae 6.
233
Pathological Report on the Histology of Sleeping Sickness and
Trypanosoniasis, with a Comparison of the Changes Found
am Animals Infected with T. Gambiense and other Trypano-
somata.
By Anton BreEINL, M.U.Dr. (Prague), J. W. Garret International - Fellow,
Liverpool School of Tropical Medicine. University, Liverpool.
(Communicated by Professor R. Boyce, F.R.S. Received April 8,—Read
May 11, 1905.)
Three cases of Sleeping Sickness and one case of Trypanosomiasis dying in
Liverpool have been histologically examined. The central nervous system of
the Sleeping Sickness cases showed the changes described by different
observers, Mott, Low, the Portuguese Commission and others. One case
exhibited an intra-pial hemorrhage of the spinal cord, extending from the
sixth cervical segment to the third thoracic segment, about 7 mm. broad. In
another case there occurred four larger hemorrhages, besides numerous
smaller ones, in the grey substance, chiefly affecting the posterior cornua and
the thoracic part of the cord. Microscopically the brain and spinal cord
showed small celled infiltration around the vessels, consisting for the most
part of lymphocytes, some plasma cells and phagocytes, between which were
a varying number of red cells in different stages of disintegration. The
intima of the vessels showed a proliferation of the endothelial cells. Red
and white blood corpuscles were often seen in the vessel walls. Here and
there the blood vessels were filled with white blood corpuscles resembling a
thrombosis.
It is most striking that the small celled infiltration is much more marked
in the grey substance of the nervous centres, especially in the large
grey ganglia, than in the peripheral parts. Very numerous capillary
hemorrhages of different sizes were present in these situations. Infiltration
around the vessels of the membranes and in the tissues of the pia and
arachnoidea was observed. Around the infiltrated vessels degeneration
of the fibres and an excess of glia cells were seen, sometimes exhibiting the
picture of red softening. The ganglia cells showed an _ irregularly
distributed degeneration, central and peripheral chromatolysis and also
partial pyknosis.
Signs of inflammation and small celled infiltration im the endo- and peri-
234 Dr. A. Breinl. On the Histology of [Apr. 8,
neurium of the peripheral nerves were seen. In two cases of longer duration
and with more pronounced symptoms of the disease, more definite changes
around the vessels of the brain and spinal cord were seen than in the third
case of shorter duration with less marked symptoms. In this case the peri-
vascular changes in the brain were small and were still less so in the spinal
cord.
In all groups of the lymph glands numerous ones were found showing the
typical appearance of hemo-lymph glands with a pronounced hyperplasia of
the connéctive tissue, a widening of the follicles and the formation of a
system of sinuses containing red blood cells and large phagocytes in a fine
threadwork of connective tissue. Others showed a transition between the
heemo-lymph glands and normal glands, one part appearing normal, the other
presenting a typical sinus formation with numerous red blood cells and
phagocytes. Nearly all the glands contained between the lymph cells a
number of blood corpuscles, many in all stages of degeneration. The spleen
was greatly congested and contained a few necrotic areas, scattered through
the organ was a little blood pigment giving the iron reaction. The bone
marrow exhibited the typical picture of red marrow with gelatinous
degeneration. The liver and kidneys showed hemorrhages between the
parenchyma cells, which latter appeared to be undergoing degeneration. In
all three cases a few large bacilli and cocci were seen which did not stain
by Gram’s method, these I consider to be due to post-mortem contamination.
The bacteriological cultivation, anaérobic and aérobic of the cerebro-spinal
fluid and the blood of two cases, did not give any growth, and moreover,
animals infected with large quantities of cerebro-spinal fluid or:blood did not
show any other symptoms than those caused through the presence of
trypanosomes in the blood.
In only one case, dying with a fair number of trypanosomes in the blood,
could I find occasionally a parasite in the congested vessels of the organs.
The one case of trypanosomiasis, which died from an intercurrent
pneumonia, did not show any other changes in the central nervous system
than the very large peri-vascular spaces, partially filled with transudate,
and sometimes containing a few white blood corpuscles. The ganglion
cells showed the changes corresponding to the hyperthermia. The lymph
glands were very hemorrhagic, some showed the typical appearance of heemo-
lymph glands.
The brains, spinal cords, ana organs of numerous animals infected with
Trypanosoma Gambiense, monkeys, rabbits, guinea-pigs, dogs, rats, and mice
were examined. One of the monkeys showed a typical hemorrhagic cicatrix
in the left lobus centralis of the brain; other monkeys and a chimpanzee
1905. | Sleeping Sickness and Trypanosomiasis, ete. 235
showed a high congestion of the vessels of the brain and spinal cord, with
hemorrhages, around the vessel walls, containing lymphocytes, a few
leucocytes, and phagocytes. The intima showed large proliferated endo-
thelial cells, the vessels often contained very many leucocytes. Numerous
hemorrhages in the grey substance of the spinal cord were frequently seen.
Some of the dogs, rabbits, and guinea-pigs showed the changes in the spinal
cord, and to a less extent in the brain. The ganglion cells exhibited similar
alteration as in the human cases. In some of the animals no changes
around the vessels and very little alteration of the ganglion cells and fibres
were noted.
Many of the lymph glands presented the picture of hemo-lymph glands
with a few pigment granules; sometimes an irregular patchy appearance was
seen, the centre consisting of a light stained area with numerous red cells
and phagocytes, the periphery of normal lymph tissue with a small number
of follicles. The spleen showed congestion in the more acute cases, with
irregular hyperplasia of the malpighian bodies, in the older cases hyperplasia
of the connective tissue. For comparison the brains, cords, and organs
of animals infected with 7. dimorphum (Gambian horse disease) were examined.
In a few cases the same hemorrhages as described above and localised in the
grey substance of the nervous centres were seen. The lymph glands showed
the peculiar appearance ; as noted above the light spaces were completely
filled with blood pigment. The spleen showed hardly any pigment.
Trypanosomes were tound mostly clumped together in the vessels of the
different organs of all animals dying with numerous parasites in the peripheral
blood.
Conclusions.
(1) In the cases of Sleeping Sickness there is a pronounced congestion of
the blood vessels of the central nervous system ‘together with a small celled
infiltration around the vessels of the brain and spinal cord, especially in the
grey substance.
(2) Chromatolysis and pyknosis of the ganglion cells of brain and spinal
cord.
(3) Inflammation of the leptomeninges of the brain and spinal cord.
(4) Neuritis of the peripheral nerves.
(5) The more chronic the case and the more pronounced the symptoms
the greater the changes in the brain and cord.
(6) The majority of the lymph glands exhibit the picture of heemo-lymph
glands.
(7) Small necroses of the spleen and signs of degeneration of the bone
marrow.
236 © Mistology.of Sleeping Sickness and Trypanosomaasis, ete.
(8) The brain of a case of Trypanosomiasis did not show small celled
infiltration.
(9) Animals infected with Trypanosoma Gambiense show sometimes
changes in the nervous system, localised in the grey matter, hemorrhages,
lymphocytes, and a few leucocytes in the peri-vascular space: hemo-lymph
glands in large nuinbers, and sometimes necrosis of the spleen and degenera-
tion of the bone marrow.
(10) Animals infected with 7rypanosoma dimorphum exhibit similar changes
in the nervous system and organs. A far greater deposit of pigment in the
lymph glands and in older cases in the spleen is present.
1905. | Sleeping Sickness and Trypanosomiasis, etc. 235
showed a high congestion of the vessels of the brain and spinal cord, with
hemorrhages, around the vessel walls, containing lymphocytes, a few
leucocytes, and phagocytes. The intima showed large proliferated endo-
thelial cells, the vessels often contained very many leucocytes. Numerous
heemorrhages in the grey substance of the spinal cord were frequently seen.
Some of the dogs, rabbits, and guinea-pigs showed the changes in the spinal
cord, and to a less extent in the brain. The ganglion cells exhibited similar
alteration as in the human cases. In some of the animals no changes
around the vessels and very little alteration of the ganglion cells and fibres
were noted.
Many of the lymph glands presented the picture of hemo-lymph glands
with a few pigment granules; sometimes an irregular patchy appearance was
seen, the centre consisting of a light stained area with numerous red cells
and phagocytes, the periphery of normal lymph tissue with a small number
of follicles. The spleen showed congestion in the more acute cases, with
irregular hyperplasia of the malpighian bodies, in the older cases hyperplasia
of the connective tissue. For comparison the brains, cords, and organs
of animals infected with 7. dimorphum (Gambian horse disease) were examined.
In a few cases the same hemorrhages as described above and localised in the
grey substance of the nervous centres were seen. The lymph glands showed
the peculiar appearance ; as noted above the light spaces were completely
filled with blood pigment. The spleen showed hardly any pigment.
Trypanosomes were found mostly clumped together in the vessels of the
different organs of all animals dying with numerous parasites in the peripheral
blood.
Conclusions.
(1) In the cases of Sleeping Sickness there is a pronounced congestion of
the blood vessels of the central nervous system together with a small celled
infiltration around the vessels of the brain and spinal cord, especially in the
grey substance.
(2) Chromatolysis and pyknosis of the ganglion cells of brain and spinal
cord.
(3) Inflammation of the leptomeninges of the brain and spinal cord.
(4) Neuritis of the peripheral nerves.
(5) The more chronic the case and the more pronounced the symptoms
the greater the changes in the brain and cord.
(6) The majority of the lymph glands exhibit the picture of hemo-lymph
glands.
(7) Small necroses of the spleen and signs of degeneration of the bone
marrow.
VOL, LXXVII.—B. 5
236 Messrs. Bateson, Saunders, and Punnett. | Dee. 1,
(8) The brain of a case of Trypanosomiasis did not show small celled
infiltration.
(9) Animals infected with Trypanosoma Gambiense show sometimes
changes in the nervous system, localised in the grey matter, hemorrhages,
lymphocytes, and a few leucocytes in the peri-vascular space: hemo-lymph
glands in large numbers, and sometimes necrosis of the spleen and degenera-
tion of the bone marrow.
(10) Animals infected with 77rypanosoma dimorphum exhibit similar changes
in the nervous system and organs. A far greater deposit of pigment in the
lymph glands and in older cases in the spleen is present.
Further Experiments on Inheritance in Sweet Peas and Stocks :
Prelimanary Account.
By W. Bateson, F.R.S., E. R. SAUNDERS, and R. C. PUNNETT.
(Received December 1,—Read December 7, 1905.)
Later results have provided expressions which include many of the peculiar
phenomena of inheritance already witnessed in sweet peas and stocks. In
sweet peas we have shown that purple may occur, as a “reversion,” from
the cross between two whites, one having long pollen grains, the other
round. Similarly in stocks, white glabrous x cream glabrous gives
“yeversionary”” F, purple hoary. (In both cases the parents are whites,
a.¢., tree from sap-colour, for cream is due to yellow plastids, recessive to
colourless plastids.)
The appearance of coloured flowers is due to the simultaneous presence
in the zygote of two factors, belonging to distinct allelomorphic pairs, which
may be spoken of as C, ¢, and R, 7, the large letter denoting presence, the
small letter the absence of the particular factor.
Hoariness of stocks is similarly due to the-coexistence of two other factors,
and the presence of either of these factors is also allelomorphic to its
absence. These two pairs are spoken of as H, h, and K,/. But, though
H and K may both be present, no hoariness is produced unless C and R,
the colour-factors, are also both present. For the actual development of
hoariness four factors are thus required. The existence of white-flowered
hoary plants creates a difficulty; but white incana is evidently a coloured
1905. | Inheritance in Sweet Peas and Stocks. 237
form in reality, for its flowers tinge on fading, and its embryo has the deep-
green colour characteristic of purple varieties. Apart from breeding-tests,
however, white hoary Bromptons show no visible indication of colour, and as
yet they constitute a marked exception.
White glabrous and cream glabrous types contain both H and K, the
two elements of hoariness. One of them contains C and the other contains
R. All sap-coloured types studied contain one only of the two factors
H, K. Consequently, we find the following result, which formerly seemed
paradoxical :—
we
1. Cream glabrous x Red or purple’ Red or purple hoary.
glabrous
2, White glabrous x Ditto Purple hoary.
3. Cream glabrous x White glabrous Ditto.
4. Any red or purple x Any red or purple Red or purple glabrous.
glabrous. glabrous.
The truth of this account appears from the fact that in F, from cream
glabrous x white glabrous all the coloured are hoary and all the whites are
glabrous. Again, purple (hoary) incana x cream glabrous gives in F, all the
hoary plants colowred, and all the glabrous plants white; while “white”
(hoary) incana x sap-coloured types gives in F, coloured hoary, coloured
glabrous, and in addition tinging “ whites” in both classes.
When a character is produced by the meeting of factors belonging to two
distinct allelomorphic pairs, the F2 ratio will be 9:7 (a2, 3+3+1), and
consequently, when in sweet peas and stocks a coloured F, is produced from
two non-sap-coloured types, the F2 ratic is 9 coloured : 7 white; but there
are 4 gametically-distinct types among the coloured and 5 among the whites.
Most of these have been now recognised experimentally.
When F; is purple the coloured class consists of purples and reds. In
both sweet peas and stocks the ratio is 27 purple, 9 red, 28 white, composed
thus :-—
4s O2eOsOs Se Seoes oil
The purples are due to the presence of a “blue” factor B, allelomorphic
to , its absence. Unless C and R are both present, B cannot be perceived
without breeding tests. The three pairs, C,c, R, 7, B, b, by entering into
all possible combinations according to the simple Mendelian system, give the
results observed.
This scheme takes no account of the sub-classes which sometimes occur
Ss 2
238 Inheritance in Sweet Peas and Stocks.
in both purples and reds. Several of these are merely superposed on the
primary classes, while others are more complex and require further analysis.
The distribution of the colours shows further complications when some
coloured strains were introduced as original parents.
“Reversion” is thus seen to be a simple and orderly phenomenon, due
to the meeting of factors belonging to distinct though complementary allelo-
morphic pairs, which at some moment in the phylogeny of the varieties have
each lost their complement.
Pollen-characters in Sweet Peas.—Gametic coupling of a novel kind exists
in this case. The whole generation in Fy, consists of 3 long: 1 round.
The whites taken alone also are 3 long: 1 round. But in the purples there
is a great deficiency of rounds, while in the reds they are greatly in excess.
This result indicates that there is a partial coupling of the long pollen-
character with the factor B, and a corresponding coupling of round pollen
with 06. This peculiarity only occurs in families which contain both
purple and red members. The gametic output of F, in these cases is
approximately
7AB+1A6+4+ 1aB+7ab,
where A is long, and a round pollen. This arrangement gives a close
approach to the observed figures :—
Purple. Red. White.
(Re ee ea Sa ee Sepa
Long. Round. Long. Round. Long. Round.
Observed ...... 1528 106 7, 381 1199 394
Calculated ...... 1448-5 122°7 122;7' 4015 12205) 407s
239
Report on the Psychology and Sociology of the Todas and other
Indian Tribes.
By W. H. R. Rivers, M.D., Fellow of St. John’s College, Cambridge.
(An Abstract of Work carried out by the aid of the Gunning Fund of the Royal
Society for the year 1901-1902. Communicated by the Secretaries of the
Royal Society. Received October 18,—Read December 14, 1905.)
Six months were spent in India, the greater part of the time being
devoted to the investigation of the Todas of the Nilgiri Hills. The senses
of these people were examined experimentally on the same lines as those
followed by the Cambridge Expedition to Torres Straits.* The general
result was to confirm the chief conclusion of this expedition that there are
no great differences between the senses of savage and civilised races. In
pure sense-acuity little difference was found, and the observations lend no
support to the view that the sense-acuity of savage or harbarous races is
superior to that of civilised man, the apparent superiority in some cases
being due to the training of observation in special directions.
In two senses only is there distinct evidence of difference between Todas
and Englishmen in sensory endowment. The Todas are distinctly less
sensitive to pain than the average educated Englishman, and they show
the same kind of deficiency in the colour-sense which has been found in
other races of low culture, especially in the Papuanft and the Egyptian
peasant.t
The Todas are distinctly less sensitive to blue than the average educated
Englishman, though differing little in sensibility to red or yellow. This
defect in the sensibility for blue is associated with the deficient nomen-
clature for this colour which is almost universal in races of low culture;
and the observations on the Todas strengthen the conclusion reached by
previous work that physiological insensitiveness is one, though only one,
of the factors upon which the defect in language depends.
The most striking feature of Toda colour-vision, however, is the great
frequency of colour-blindness. About five hundred individuals were tested,
and over 12 per cent. of the males were found to suffer from typical red-
green blindness, the proportion in European races being about 4 per cent.
In most races of low culture colour-blindness is less frequent than in
* ‘Reports of the Cambridge Anthropological Expedition to Torres Straits.’ Cambridge,
vol. 2, Part I, 1901, and Part II, 1903.
t Loe. cit., p. 48.
t ‘Journ. Anthrop. Inst.,’ 1901, vol. 31, p. 229.
240 Psychology and Sociology of the Todas, ete.
Europe, but the Todas show the highest recorded frequency of this condition
in any race. By means of the genealogies preserved by the Todas the
relationship between the colour-blind people could be traced, and a body of
material was obtained which illustrates the mode of hereditary transmis-
sion of the defect. .
The Todas were found to be subject to various geometrical-optical illusions ;
and quantitative observations were made on the illusion of compared vertical
and horizontal lines and on the Miiller-Lyer illusion. The Todas are
subject to the former in a greater degree than English observers, and to
the latter in a smaller degree. The two illusions differ in nature: the
former is probably largely physiological in origin, and is neutralised by
the experience of civilised life, while the latter is more strictly psycho-
logical in character; and the different reaction of the Todas to the two
illusions is in accordance with this difference in their nature.
In every measurement the degree in which the individuals of each
race differed from one another was studied; and a mass of material was
collected for the study of variability in the reaction to psychological tests,
and for the analysis of the complex conditions upon which the coefficients of
variation depend.
On comparing the observations of Todas, Papuans, and Englishmen, all
tested by the same methods, and chiefly by the same experimenter, it is
found that there is some evidence of a correlation between the degree of
general intellectual development and certain simple mental properties or
activities which can be tested by experimental methods. In general
intellectual development the Todas occupy an intermediate position between
Papuans and Englishmen, and a similar intermediate position is occupied by
them in connection with many of the tests.*
The social and religious institutions were also studied. The sociology was
investigated largely by means of the genealogical method,f and the system
of kinship, the complex marriage regulations and the laws of inheritance
and property were worked out in detail.
The Todas were found to possess a highly elaborate religious ceremonial
of which only brief sketches had previously been published, while many
ceremonies had wholly escaped observation. In consequence, much time
was devoted to the detailed investigation of this ceremonial and of the other
features of the Toda religion. Evidence is given that this religion is one
which has undergone degenerative changes, and some evidence is advanced
* A full account of the senses of the Todas will be published shortly in the ‘ British
Journal of Psychology, vol. 1, Part IV.
+ ‘Journ. Anthrop. Inst.,’ 1900, vol. 30, p. 74.
Nitrification with reference to the Purification of Sewage. 241
in favour of a view that the Todas are a people who have once had a culture
higher than that they now possess. When the customs and institutions of
the Todas are compared with those of other parts of India, it is found that
there is most resemblance with the people of Malabar; and the view is
advanced that the Todas migrated to the Nilgiri Hills from Malabar, and are
possibly allied in race to the two chief castes at present existing in that
district, the Nairs and Nambutiris.
In addition to the work on the Todas, observations were also made on
members of other tribes. The vision of the Sholagas and Uralis, two wild
jungle tribes, was investigated* from several points of view; and observa-
tions, chiefly on colour-blindness, were made on members of other castes or
tribes.
A Study of the Process of Nitrification with reference to the
Purification of Sewage.
By Harrrerre Cuicx, D.Sc.
(Communicated by Professor H. Marshall Ward, F.R.S. Received April 1,—Read
May 11, 1905.)
Introduction —That nitrification is a biological process was first established
with certainty, after long controversy, in 1888, by the decisive experiments of
Platht and Landolt, who in this matter confirmed the previous researches
of Schlésing and Muntz,§ Warington|| and Soyka.{
The discovery of the active living agents followed soon after, when
Winogradsky** (1890 to 1892) isolated the two sets of organisms which, as
he showed, co-operate to produce natural nitrification. These were (1) the
nitrite-producer, b. nitrosonvonas, which oxidises ammonia to the nitrite stage
only, and (2) the nitrite-producer, &. nitrobacter, which carries on the
* ‘Bull. Madras Government Museum,’ 1903, vol. 5, p. 3.
+ Plath, ‘Landw. Jahrbiicher, v. H. Thiel, vol. 16, hft. 6, and ‘Centralbl. f. Agri-
kulturchem. v. Biedermann,’ vol. 17, 1888.
t Landolt, ‘Deutsch. Landw. Presse, vol. 15, and ‘Centralbl. f. Agrikulturchem., vol.
17, 1888.
§ Schlésing and Muntz, ‘Comptes Rendus,’ vols. 84 and 85, 1877, and vol. 89, 1879.
|| Warington, ‘Journ. Chem. Soc., vol. 33, 1878, and ‘Landw. Versuchsst.,’ vol. 24,
1880.
4 Soyka, ‘ Zeitschr. f. Biologie, vol. 14, 1878.
*= Winogradsky, ‘Ann. de l’Inst. Past., vol. 4, 1890, and vol. 5, 1891; also ‘ Archives
des Sci. biol. de St. Petersb.,’ vol. 1, 1892.
242 Dr. H. Chick. The Process of Nitrification [Apr. 1,
oxidation to nitrate but cannot act upon ammonia, being indeed inhibited mn
its development by minute traces of that substance. Winogradsky, by him-
self, and in conjunction with Omeliansky,* subjected these bacteria to a very
exhaustive study. The most striking characteristic that they demonstrated
was the marked repugnance of both forms to organic substances. Not only,
in opposition to the rest of the plant world, do these organisms make no
nutritive use of sugars, peptones, etc., but the presence of more than a trace
of such organic substances was found to entirely inhibit their development,
thus explaining the failure of all attempts to isolate these bacteria by using
the ordinary nutrient culture-media:; Winogradsky, on the other hand, had
succeeded in cultivating them by employing a silica-jelly-medium impregnated
with inorganic salts, and a total imability to grow on organic nutrient media
was afterwards put forward by him as a definite practical criterion of the
purity of cultures of nitrifying bacteria. This criterion has been challenged
by Burri and Stutzer,t by Stutzer and Hartleb,t and later by Fremlin.§
It has been shown by Winogradsky,| and also by Girtner, Frankel, and
Kriigerf that the former workers were misled by an admixture of non-
nitrifying organisms. The most recent work, that of Boulanger and
Massol,** and of Wimmer tf confirms Winogradsky’s criterion.
The special case of nitrification considered in this paper is that occurring
during sewage purification, which aims at the complete oxidation and
mineralisation of putrescible substances present. Nitrification is here of great
importance, and the effluent of perfectly-treated sewage should contain all its
nitrogen in the form of nitrates.
Although land-treatment of sewage is theoretically the most economical,
yet artificial processes, by which space can be saved, have often to be employed.
Two processes concern us here, both involving the use of “filter-beds” of
coke or other porous material, in which the sewage, usually after having
been treated in a “septic tank” is oxidised by bacteria. (1) Contact Filters.
—In these the filter-bed is first entirely filled up with the liquor and then
after a time allowed to empty slowly, and finally to remain empty for a
period. This cycle usually occupies about eight hours, and often may have
to be repeated before the effluent is sufficiently purified. (2) Continuous
* Winogradsky and Omeliansky, ‘Centralbl. f. Bakt.,’ 2 abt., 5, 1899.
+ Burri and Stutzer, ‘Centralbl. f. Bakt., 2 abt., 1 and 2, 1895 and 1896.
} Stutzer and Hartleb, ‘Centralbl. f. Bakt.,’ 2 abt., 2 and 3, 1896 and 1897.
§ Fremlin, ‘Journal of Hygiene,’ vol. 3, 1903.
|| Winogradsky, ‘ Centralbl. f. Bakt.,’ 2 abt., 2, 1896.
4 Gartner, Frinkel, Kriiger, ‘Centralbl. f. Bakt.,’ 2 abt., 4, 1&98.
** Boulanger and Massol, ‘Ann. de l’Inst. Past.,’ vol. 17, 1903.
tt Wimmer, ‘ Zeitschr. f. Hygiene,’ vol. 48, 1904.
1905. | with reference to the Purification of Sewage. 243
Filters.—In this procedure the liquor trickles continuously through the filter-
bed, being uniformly distributed by sprinklers, while as perfect aération as
possible of the bed is maintained.
The objects of the present research were mainly the following :—
1. The detailed chemical study of the course of the nitrification occurring
during the filtration of sewage, especially during the maturing period of the
filter, and the comparison of the “contact ” and “continuous” methods
(Section I).
2. The isolation and study of the organisms concerned, and comparison
with those isolated from the soil by Winogradsky. The amount of organic
matter accumulated in a sewage filter is comparatively great, and it seemed
most unlikely that nitrification should here also be the work of bacteria so
extremely sensitive to the presence of organic matter. One seemed com-
pelled to believe that other and different bacteria must be here engaged
(Section II).
3. The study of the question of absorption of ammonia upon the surface
of filtering material previous to nitrification (Section III).
These researches were begun in Vienna in 1901, and were resumed in
Munich in 19035, after a break of two years. I am very happy to have this
opportunity of thanking Professor Max Gruber for his kind hospitality
extended to me in the hygienic institutes of both cities, as well as for the
valuable advice and kind assistance he constantly gave me in the course of
the work. My thanks are also due to the Royal Commission on Sewage Dis-
posal for granting me leave of absence in 1903 to continue the research
in Munich. I should also add that part of the expense of the work was
defrayed by a grant from the Royal Society.
Section 1—Chemical Study of Nitrijication in Experimental Filters.
Description of Apparatus and Methods of Analysis—Small experimental
filters were erected, consisting of glass cylinders 50 em. high and 12 em. in
diameter ; these were placed one above the other, fitted well together by
means of specially ground rims, and covered on the outside with black
glazed paper. There were altogether three filters, differing only in height—
200 cm., 100 cm., and 50 cm. respectively. Fig. 1 is a diagram of the filter
of medium height, showing the arrangements made to allow of samples being
drawn off, and of the temperatures being measured at different depths; the
tall filter, consisting of four cylinders, had the three upper ones similarly
constructed. The filters were filled with small coke, carefully sifted and ot
a uniform size (mean diameter 3-5 mm.). By volumetric measurements with
244 Dr. H. Chick. The Process of Nitrification | Apr. 1,
water, it was found that when this coke is packed into a space, the volume of
Fic. 1—Diagram of the “ Con-
tinuous” Filter, of medium
height, showing construction.
The contour of the filter is
represented as interrupted at
K, to show the arrangement
of the thermometer and the
collecting funnel.
the interspaces between the pieces is 35 per cent. ;
the volume of the pores inside the pieces (amount
of water retained on draining) is 20 per cent.;
and the volume of solid coke is 45 per cent. of
the whole space. ‘The three filters were fixed to
the wall, near together, and all treated in exactly
the same way, 7.¢., as continuous filters. Each
received 4 litres of liquid daily, and the sewage
employed was the liquid manure (“Jauche”)
from a neighbouring cowshed; this proved a
very suitable material, after a rough filtration
through glass wool, and dilution to 1 in 20 with
tap-water.
This liquid was contained in a 10-litre reservoir
bottle of the Mariotte type, from which it”
dropped regularly into a small vessel containing
a siphon arranged to empty when 100 c.c. had
collected (A, fig. 1) into a sprinkler (B, fig. 1),
the object of which was to distribute the liquid
as evenly as possible over the surface of the
filter.
The methods of analysis employed in following
the course of the oxidation of nitrogen were
those usually adopted in such work,* but the
following details may be given :—
In the estimation of free and saline ammonia, 1 to
10 e.c. of the liquid was taken and diluted in a retort
with about 500 c.c. NH;-free water, and distilled, it
being found unnecessary to add any alkali Three
successive portions of 50 ¢.¢. were distilled off and the
ammonia they contained estimated by means of Nessler’s
reagent. Albuminoid ammonia was afterwards estimated
by adding to the same retort a definite amount of
“alkaline permanganate solution,” distilling as long as
ammonia came over in the distillate, and estimating
these amounts in the same way.
Oxidised nitrogen in the filtrates was detected by
means of the reaction with diphenylamine sulphuric acid.
Nitrites were distinguished by reactions with acidified
* Cf. ‘ Report of Royal Commission on Sewage Disposal,’ vol. 4, part 5.
+ ‘Sew. Com. Report,’ vol. 4, part 5, appendices 3 and 4.
1905. | with reference to the Purification of Sewage. 245
starch-zine-iodide solution, and with metaphenylene-diamine, and estimated by the use of
the latter. When both nitrites and nitrates were present, they were estimated together by
the indigo method (Tiemann-Giirtner’s “ Wasseranalysec”) and the figure for nitrates
obtained by subtraction. This method was afterwards given up in favour of the copper-
zine couple method,* where, to allow for. traces of ammonia originally present in the
solution, or introduced during analysis, a control estimation was ulways made ; this control
was carried out in every way like the real analysis, except that no couple was introduced,
and the ammonia obtained was subtracted from that found in the actual estimation.
Total nitrogen was estimated in the sewage by Kjeldahl’s method, a small amount of
Na,SO, only being added during the preliminary heating.
The oxidisability (‘‘ Oxidirbarkeit,” or measure of oxidisable substances present) was
estimated by reduction of permanganate in alkaline solution on boiling for ten minutes,
care being taken to keep the external conditions (such as concentration, size of flask, total
amount of liquid present, temperature, and time of reaction) constant in all determina-
tions. Periodic examination of sewage and filtrate in this way gave useful comparative
results and a means of following the course of the general oxidation.
Cowrse of General Oxidation During Maturation: First Appearance of
Oxidised Nitvogen.—Vhe filters were started on February 20, 1901, and their
action carefully controlled by means of analysis from that time onwards.
Especial attention was paid to the period of maturation from the time of
first using to that of full efficiency, as it was thought this should throw
light generally upon the manner of their working. The sewage employed
contained on an average :—total nitrogen 10 parts, organic matter (by
evaporation and ignition) 21 parts; oxidisability (expressed in terms of O)
about 11 parts per 100,000 by weight. The course of the oxidation will
be seen by reference to Tables Ia, Ip and II. On March 7 (see Table II,
analyses 1 to +) there was a marked amount of general oxidation taking
place in all three filters, or at least a reduction of oxidisable substances, but
there was no trace of oxidised nitrogen in the filtrates nor was there any
diminution in ammonia. Ovxidised nitrogen first appeared.in the tall filter
on March 18 (the filtrate being then clear, bright and without smell), three
days later it was detected in the medium filter and seven days later in the
short filter. During this first period (after starting and before the occurrence
of nitrification) the amount of ammonia in the sewage was frequently
compared with that in the filtrates and found to be always the same.f On
March 7 this was proved to be so for all three filtrates, and even on
March 28, when the first trace of oxidised nitrogen appeared in the filtrate
from the short filter there was no diminution of the free and saline ammonia
coming through (¢f. analyses 10 and 13). So, generally, Tables II and IIT
* Sutton, ‘ Vol. Analysis,’ 8th ed., p. 452.
+ As the sewage varied considerably in composition from time to time, care was always
taken that the sewage and filtrate analysed for comparison should correspond to one another
as nearly as possible. i
246 Dr. H. Chick. The Process of Nitrification | Apr. 1,
seem to show that loss of ammonia goes hand in hand with production of
oxidised nitrogen.
Tables IA (Vienna) and Isp (Munich) give a clear idea of the general
progress of the oxidation of nitrogen. They are compiled from analyses
made from time to time, and while the times given for the later stages are
approximate, those for the first stage are exact, being based on almost daily
tests.
The Nitrite Stage—After the first appearance of oxidised nitrogen in the
filtrates, nitrification went ahead, and, in the case of the tall filter, five days
later there was nitrous nitrogen in the filtrate about equal in amount to the
total nitrogen going on. The two stages in which nitrification occurs were
well separated in time, and show very distinctly, first, the production of
nitrites in quantity without nitrates, and finally the complete oxidation to
nitrates, nitrites being absent. For example, in the case of the tall filter,
five weeks after having been started (Table II, analyses 10 and 11) the
sewage contained 17 parts ammoniacal and albuminoid nitrogen, which in
the filtrate was reduced to 1:5 parts, while 10 parts nitrogen were
present as nitrites, nitrates being altogether absent. Three days later a
similar result was obtained (analyses 14 and 15), eight weeks later nitrates
were being formed in small amount, and an analysis of the filtrate made four
months after starting (analysis 21) showed a complete oxidation of the
nitrogen, nitrates being present in quantity unaccompanied by nitrites. In
the case of the short filter the process was much slower, for an analysis
made after four months showed production in the filtrates of nitrites only
(analysis 23). This comparative lack of efficiency may be referred to the
lower temperature* of the short filter, for an analysis on the same date of
the liquid from No. 1 tap of the tall filter, 50 cm. from the top (Table III,
6 and 7) showed the presence of nitrates in abundance, and only traces of
nitrites.
* An exactly parallel fact was noticed with regard to the time of the first appearance of
oxidised nitrogen in these two filters. Strictly speaking, the top section of the tall filter
should have been exactly comparable with the short filter, and the only possible
explanation is that, as the first division of the latter filter stood 150 cm. higher in the
room, the discrepancy was due to a temperature difference. Regular observations of
temperature had been made by means of the thermometers in the filters, and the higher
position in the room was found to be constantly from 1°5° to 2°5° C. higher than the lower
one, the temperature of the interior of the filters differing hardly at all from that of the
surrounding air. This explanation is confirmed by the results of the Munich experiment,
where all the six filters were arranged so that their tops, and not their bottoms, were on
one level ; hence the first divisions of all were at a similar temperature, and it was found
that the first oxidation of nitrogen was observed after the same period in all the three
filters of each set.
1905. | with reference to the Purification of Sewage. 247
This complete separation of a nitrite from a nitrate stage is doubtless due
to the comparatively strongly ammoniacal nature of the sewage employed.
Previous observers*f have shown the inhibitive effect of ammonia upon
nitrate-production, and it is probable that during the earlier stages of the
maturing period the nitrate bacteria were unable to become established in
the filter, and only later, when the ammonia of the sewage was being rapidly
oxidised to nitrites, was the environment suited to their growth and develop-
ment. The sewage employed frequently contained more than 15 parts free
and saline ammonia per 100,000, a concentration which has been shownf to
be sufficiently high to check completely the production of nitrates in pure
culture. An interesting confirmation of this explanation was obtained in the
maturing of the Munich filters, where nitrates appeared in the filtrates very
soon after the first appearance of nitrites. There was here no such “ nitrite
stage,” and the sewage was much less ammoniacal (2 to 4 parts ammoniacal
nitrogen per 100,000).
Difference of Function in Different Strata of the Filters—An attempt was
made to study the course of the oxidation at different depths in the tall and
medium filters (see Table III). In analysis 3 the “ oxidisability ” was taken as
a criterion and the decrease in the first 50 cm. of the tall filter was found to be
almost as great as in the whole length of the filter. One may therefore suppose
that the mechanical deposition of suspended particles as well as the absorption
of the more complicated organic matter in solution takes place principally in
the upper layers of the filter. It is also apparent from analysis 4 that the
formation of nitrites (these analyses were made during the nitrite stage) did
not at that date take place in quantity in this upper layer but lower down
for the most part; this same fact is also shown in analysis 2. In the latter
case the free and saline ammonia was also estimated, and the decrease, which
is so marked as the sewage passes through the filter, was found not to begin
until after the first 50 cm. were passed. The same phenomenon appears in
analysis 8 and is a striking instance of the principle, already alluded to, and
discussed at length in the section devoted to absorption, that the dis-
appearance of ammonia and the oxidation of nitrogen are closely associated
both in time and space.
Comparison of Contact and Continuous Filters—Munich Experiments —
From the mature Vienna filters attempts were made to isolate the nitrifying
organisms, but before much progress had been made the work was dis-
continued and was not again resumed until after two years. This second
time, in Munich, in 1903, fresh filters had to be matured, and a second
* See footnote **, p. 242.
+ Warington, ‘Chem. Soc. Journ.,’ vol. 35, 1879, and vol. 59, 1891.
248 Dr. H. Chick. The Process of Nitrification [Apr. 1,
opportunity was afforded for studying the maturing process, and nitrification
generally.
Filters like those previously described were again erected, and three of
them treated as formerly with a continuous trickle of diluted liquid manure.
Another similar set of three filters was treated, for contrast, as contact
filters by the procedure mentioned on p. 242. Eight litres of sewage were
treated every 48 hours by each of the filters. The capacities of the tall,*
medium and short filters were respectively, after wetting, 6, 4 and 2 litres, so
that each filling remained in contact at least four hours in the two taller filters
and two hours in the short one. For 38 out of the 48 hours of the cycle the
filters were empty oremptying. ‘These contact filters were cone-shaped below,
with a narrow opening that could be closed with a cork for the purpose of
filling them.
In the case of the continuous filters, oxidised nitrogen again made its first
appearance in the filtrates four weeks after starting (compare Tables JA and
1s). The fact that all three Munich filters behaved alike in this respect
(forming a contrast to the Vienna ones) has already been explained as a
temperature effect, see p. 246. It was noticed that during the maturing
period the two different stages of nitrogen oxidation merged one into the
other, and were not so clearly separated as was the case in the Vienna filters
(Tables LA and Is); noticeably there was here no long period in which nitrites
were formed in quantity without any accompanying nitrates. This difference
has already been discussed (p. 247), and explanation is doubtless to be found
in the much less ammoniacal nature of the sewage here employed.
The contact filters did not yield nearly such good results as the continuous
filters (Table Is). The period which elapsed before nitrogen oxidation was
apparent was, in the former, more than half as long again as in the latter.
Again, when the short continuous filter showed complete oxidation of its
nitrogen, the tall contact filter still showed presence of nitrites im its filtrate. —
The Munich continnous filters had completely matured in about ten weeks
from the time of starting, but they were yielding a very satisfactory effluent
much earlier. After three months the sewage was changed for a much
more strongly ammoniacal liquid (cows’ urine, diluted 1 in 100, containing
14 to 17 parts ammoniacal nitrogen per 100,000), in order to test the
capabilities of the filters as regards nitrogen oxidation. Most satisfactory
results were obtained (Table II, analyses 33 to 56, and Table IV), the
filtrates contained, as a rule, only traces of ammonia and nitrites, but
abundance of nitrates. Attempt to further tax the capabilities of the filters
* Tn these Munich experiments the tall filters, both contact and SERENE OS, were only
150 em. high, instead of 200 cm., as in Vienna.
1905. | with reference to the Purification of Sewage. 249
met with failure. Cows’ urine diluted only 1 in 50, and containing about
30 parts ammoniacal nitrogen per 100,000, was put through the medium
filter for about a fortnight, and also at a later date through all three filters.
but it was found that they were incapable of oxidising so concentrated
a liquid, and the quality of the filtrates deteriorated (¢7: Table IV).
Throughout the history of these filters there was a considerable loss of
total nitrogen from the sewage while filtering through, but it was specially
noticeable during the period when the diluted urine was being treated, when
in some cases not much more than half the original nitrogen was present in
the filtrate (Table II, analyses 33 to 56). This loss is doubtless due to an
escape of free nitrogen, set free possibly by decomposition of ammonium
nitrite, a very probable intermediate product in the nitrification of ammonia
(NH,NO, = 2H20 + N2). This loss of nitrogen was not so marked in the
ease of the Vienna filters (Table II, analyses 1 to 25), though it occurred later
to some extent. These differences are probably due to the absence or
presence in quantity of the organisms involved.
The Munich continuous filters in their later history, and they were worked
for about a year, possessed an efficiency rarely met with in large scale filters,
showing that this type can give excellent results in the absence of much
suspended matter.* The larger filters could not be considered to be heavily
worked, but the short filter, which had a capacity of two litres and treated
four litres of sewage daily, approximated more nearly to a practical installa-
tion. It oxidised daily about 0°5 gramme nitrogen, and this resuit must be
considered extremely satisfactory when the high nitrogenous concentration
of this special sewage (14 to 20 parts of ammoniacal nitrogen per 100,000
instead of the 3 to 8 parts usual in ordinary sewage) is kept in mind. The
quantities of nitrate appearing in the filtrates from these filters have rarely,
if ever, been obtained in practice on the large scale.
Section 11.—Bauciteriological Investigations.
Enumeration of Nurifying Bacteria in the Filtrates—It was thought
worth while to attempt to count the numbers of nitrifying bacteriat present
in the filtrates from the filters, as it was conceivable that such enumerations
might furnish a bacteriological criterion of the quality of sewage effluents.
The practical utility of this procedure is, however, diminished by its
* This can be removed in practice by a preliminary screening or septic tank treatment.
+ The recent work of C. C. Frye (‘Report of Roy. Com. on- Sewage Disposal,’ vol. 2,
1902, p. 9) has experimentally verified the view which has been generally held, though
doubted in some quarters, that all the nitrification taking place in sewage filters is the
work of hving organisms, and none of it purely chemical.
250 Dr. H. Chick. The Process of Nitrification [Apr. 1,
slowness, due to the sluggish growth of the organisms and to the extremely
small numbers of them introduced in the higher dilutions. The method
might, perhaps, give useful comparative results even without allowing the
maximum time for development in the subcultures, and the rate of growth
could be accelerated by a temperature of 28° to 30°C.
The enumerations were made as follows :—The filtrate was successively diluted with
sterile water to a tenth degree six times ; of these six dilutions (viz., 1 in 10, 1 in 100,
1 in 1000, 1 in 10,000, 1 in 100,000, 1 im 1,000,000) 1 ¢.c. was used in every case for
inoculation into bouillon and into Winogradsky’s ammonia and nitrite-containing media
respectively,* which were distributed in test-tubes, each containing about 5 ¢.c. The
ammonia tubes were subsequently tested for production of nitrites with acidified starch-
zinc-iodide solution, and the nitrite tubes for nitrates with diphenylamine sulphuric acid
(after evaporation to dryness with NH,Cl if any nitrite remained unoxidised).
The numbers in which the nitrifying bacteria are present are surprisingly
large (see Table V), and it will be seen that there is no strict relation
between the numbers present respectively of nitrite and nitrate producers ;
but the latter would appear to be present generally in less amount even
when the filtrate shows complete oxidation of its nitrogen to nitrates. The
filtrates used were in all cases from the Munich continuous filters.
Isolation of the Nitrite Producer—tThe isolation of a nitrite-producing
bacterium in pure culture was found to present considerable difficulty and
many unsuccessful trials were made. Attempts were first made to isolate it
directly from the coke of the filters by the method of dilutions. This
method, originally invented by Lister, was formerly employed by Waringtont
and P. and G. Franklandt for the same purpose, but with only partial
success. The method here employed was similar to that used by the
Franklands, except that much higher dilutions were made, and a larger
number of tubes (about 200) containing appropriate culture media§ were
sown with small amounts of liquid from the higher dilutions, bouillon tubes
being also similarly inoculated as controls. Repeated attempts to isolate
from the coke of the filter were only partially successful.|| A culture
was, however, obtained which was comparatively, but not absolutely, pure ;
this was used for further isolation experiments, and will be referred to as
eulture “a.”
* Omeliansky, ‘Centralbl. f. Bakt.,’ 2 abt., 5, 1899.
+ See footnote, p. 247.
{ P. and G. Frankland, ‘ Phil. Trans.,’ B, vol. 181, 1890.
§ Culture solutions used were diluted urine and Winogradsky’s solutions.
|| Starting from such very impure material, the dilution method does not give an
adequate return for the great labour it entails. The filtrates might have proved better
original material, as in them the nitrifiers sometimes predominated (see Table Y).
1905. | with reference to the Purification of Sewage. 251
Culture “a,” and all cultures showing production of nitrites* invariably
contained in quantity a small oval bacillus or coccus, which was recognised
as the nitrite-producing organism.
Attempts to obtain a pure culture were further made with the use of
ammonium agar as medium,t but without success. The plate cultures
showed vigorous formation of nitrites,* but all nitrifying subcultures were
found to be impure. The employment of a similar medium composed of agar
and diluted cows’ urine was equally unsuccessful.
Ordinary gelatine plate cultures were made and bouillon was inoculated
from the impure cultures; none of the colonies separated from the former
were able to nitrify, although 40 were investigated. From the growths in
bouillon, plate cultures were also made on nutrient gelatine and agar, and
70 of the organisms separated were further investigated, but in no ease did
nitrification occur. This seemed to show that the nitrifying organisms
in filters resembled those of Winogradsky very closely. Therefore, in order
to decide if the nitrifying organism was or was not able to live in the
bouillon, an ammoniacal medium was directly inoculated from the growths
in bouillon. Usually there was no nitrite-production (¢g., Table VI,
culture “a”), and indeed the oval bacillus could in no case be traced in
the bouillon growths. In one instance, however (Table VI, culture “d”),
inoculation from a bouillon growth led to nitrification, but this property was
lost after a second generation in bouillon; it therefore seemed probable that,
if the nitrifier had not been killed in the bouillon, it certainly had not been
able to multiply there.
It thus was evident that, contrary to expectation, the nitrite-producing
organisms of sewage filters were also unable to grow upon media containing
organic matter; recourse was then had to silica plate cultures, which were
made and inoculated according to the directions given by Omeliansky.t
This operation was accomplished with comparative ease if the original
sodium silicate was quite pure; the study and isolation of the separate
colonies was, however, found to be exceedingly difficult. The sub-cultures
* The test for production of nitrites was usually made by allowing a little of the
culture fluid, withdrawn with a sterile pipette, to drop into a small quantity of acidified
starch-zinc-iodide in a porcelain dish. This was preferred to the similar test with
diphenylamine, partly because of its specific nature, and partly because the ferric salt
present in the sediment of the culture-tubes also yielded a slight blue colour with
diphenylamine.
+ (NH4)2S0,, 2:0 gr. ; NaCl, 20 gr. ; K2HPO,, 1:0 gr. ; MgSO,, 0°5 gr. ; MgCO,, in excess ;
agar-agar (purified by washing, Beyerinck’s method, ‘ Centralbl. f. Bakt.,’ vol. 19, 1896),
20 gr. ; distilled water, 1 litre.
{ See footnote, p. 250.
VOL. LXXVII.—B. T
252 Dr. H. Chick. The Process of Nitrification [Apr. 1,
obtained were to all intents and purposes pure cultures, showing pure
pictures of an oval, almost spherical organism, resembling the nitrosomonas
of Winogradsky, except that it seemed to be somewhat smaller in size.
It appeared constantly in the form of zoogloea embedded in the particles of
magnesium carbonate at the bottom of the culture tubes, and it stained
easily and well. The individual bacteria were often found to be well
separated in a culture, but an actively motile stage was not observed.
These cultures, however, still gave a growth, though extremely slow, in
bouillon, and this consisted of the other quite inconspicuous organisms
present. By means of the dilution method, pure cultures were obtained
which yielded absolutely no growth in bouillon when preserved indefinitely
either at 37° or at the room temperature. These pure cultures were not,
however, robust, and they nitrified very feebly; attempts are now being
made to obtain vigorous pure cultures.
Isolation of the Nitrate-producer.—The dilution method was also employed
for the isolation of the nitrate organism, the original material being a
culture obtained during the enumeration experiments (Table V), which
showed active oxidation of nitrites. A culture was separated which consisted
of the nitrate bacterlum mixed with one other species, and the combination,
referred to in future as culture “d,’ formed a very interesting symbiosis.
Pure cultures were obtained from culture “d” by making surface plate
cultures, in great dilution, on nitrite agar.* These pure cultures showed a
small non-motile bacterium, agreeing in essentials with Winogradsky’s
organism, though somewhat larger in size. It was a bacterium very thick
in comparison with its length, so that it often appeared to be almost a
coccus; stains were badly taken up, and it frequently appeared imperfectly
and irregularly stained. These pure cultures rapidly changed nitrite to
nitrate, when growing in nitrite-containing medium,} the nitrite present
being sqmetimes completely oxidised in less than two weeks. Bouillon on
the other hand remained indefinitely sterile; the tubes were kept under
observation for seven weeks without there being any sign of growth.
* Omeliansky, ‘ Centralbl. f. Bakt.,’ 2 abt. 5, 1899.
+ Winogradsky’s nitrite culture solution was invariably employed, and the cultures
were tested from time to time for the production of nitrates. When time enough had
elapsed and all nitrite had disappeared, then, on testing the culture liquid, a negative
reaction with starch-zinc-iodide, and a positive with diphenylamine proved the presence
of nitrates. But if all nitrite were not oxidised, the remainder was decomposed by
evaporating to dryness with a little NH,Cl, and the residue dissolved in water and tested
for nitrates with diphenylamine. This method has been shown to be quantitative when
such substances as sugar and peptone are present (Frankland, ‘ Journ. Chem. Soc.,’ 1888),
and it is possible it might also prove a useful method of estimating nitrates in presence of
nitrites in sewage effluents.
1905. | with reference to the Purification of Sewage. 253
Pure cultures were also obtained with more difficulty directly from less
pure material, by means of nitrite agar plates, but the organism isolated
was in every case the same.
All attempts to isolate a nitrate organism by means of ordinary nutrient
agar and gelatine were unsuccessful. In no instance was nitrite oxidised
to nitrate by any organism separated on such plate cultures, though over
40 such organisms were investigated.
Experiments with “ Symbiotic” Cultures of the Nitrate-producer.—Although
nitrobacter, when alone, is incapable of growing in bouillon, it would appear
to be capable of surviving an inoculation into bouillon if not alone, but
growing with certain other bacteria. A very instructive set of experiments
was made with culture “d” (Table VII), in which this strain was inoculated
into bouillon through four generations. From each set of tubes nitrite
medium was inoculated, and it was found that the change to nitrate
occurred invariably in the tubes sown from the earliest bouillon generation,
and in two instances also from those sown from bouillon of the fourth
generation (Table VII, d3 and ds). The quantities inoculated were large, one
or two drops, but it is impossible to believe that nitrobacter would still be
present in a fourth generation if no multiplication had taken place in the
bouillon. Pure cultures of the uitrate-producer showed no such effects ;
inoculated bouillon remained quite clear; examined under the microscope it
showed complete absence of bacteria, and nitrite tubes inoculated from the
bouillon in no case showed any oxidation to nitrate. In Table VIII are
shown the results of further experiments in which three pure and four mixed
cultures were compared in this respect, and one is compelled to conclude, in
explanation, that the presence of the accompanying organism in some way
protects the nitrate bacterium from adverse influences present in the bouillon,
which it is unable to withstand if alone. Without further experiment, any
attempt to explain in what this action really consists must be pure conjecture,
but it is possible that the harmful organic substances present are in some way
altered by the accompanying organism, and it would be interesting to see
whether the nitrate organism in pure culture could thrive in bouillon
previously exhausted by its companion.*
Phenomena which present an interesting analogy with these observations
are found in the case of certain anaerobic organisms, one instance of which
has been precisely investigated by Winogradsky, viz. that of Clostridium
Pasteurianum.t This strictly anaerobie species was found to be capable of
* The experiment was not made in this instance because the cultures had then been
isolated a considerable time and their properties were enfeebled.
+ ‘ Archives des Sciences biolog. de St. Petersb.,’ vol. 3, 1895.
254 Dr. H. Chick. The Process of Nitrification [Apr. 1,
growing aerobically when, and only when, associated symbiotically with a
certain aerobic organism which removed the surrounding oxygen and created
an oxygen-free environment for it. Such symbioses of various grades must
be frequent in Nature where the “pure culture” is almost unknown. The
part played by the artificial pure culture in the progress of bacteriology has,
of course, been enormous, yet its possibilities are limited, and one must look
to the investigation of regulated simple symbioses for a nearer approach, in
the laboratory, to the workings of Nature.
A break of two years occurred during the course of these investigations.
After they were again resumed, Dr. Schultz-Schultzenstein* published the
results of bacteriological investigations, having the same aim as the present
work. He isolated two kinds of nitrifying organisms from the material
of coke sewage filters at Karolinenhéhe, near Charlottenburg, which
corresponded exactly to those isolated from the soil by Winogradsky, and
no other nitrifying organisms were found. His researches must be regarded
as the first published successful attempt to investigate the organisms
concerned with nitrification during the artificial purification of sewage, and
the results are entirely confirmed by the present investigation. In spite
of this anticipation of my identification of these bacteria, I have thought
it worth while to describe my isolation experiments in detail, because in a
subject of such technical difficulty the experience of an independent worker
may be of use to others.
Section III.—Absorption of Ammonia and Ammoniacal Compounds during
Sewage Purification.
It has been held that a most important preliminary to nitrification, both
in the soil and in sewage filters, is to be found in an absorption of ammonia
and ammonium compounds upon the surface of the particles of soil or of
filtering material respectively. In the case of the soil, a long controversy
has taken place as to whether a physical or a chemical process was here in
question, and the former view, maintained notably by Liebigt and his school,
has on the whole prevailed. This “adsorption” of ammonia plays an
important part in the current doctrine of the action of sewage filters, which
considers that nitrification could not take place in the short time taken by
* Schultz-Schultzenstein, ‘Mitt. a. d. Kon.-Priifungsanst. f. Wasservers. u. Abwas-
serbeseit,’ 1903.
+ Way, ‘Agric. Soc. England Journ.,’ series 1, vols. 11—13, 1850—1852, and Mayer,
‘Lehrbuch der Agrikulturchemie,’ 1871. Lemberg, ‘Zeitschr. d. deutsch. geol. Gesellsch.,’
vol. 28, 1876.
{t ‘Liebig, ‘Ann. Chem. Pharm.,’ vol. 94, 1855, aud vols. 105 and 106, 1858.
1905. | with reference to the Purification of Sewage. 255
the liquid to pass through the filter, and that the nitrites and nitrates
appearing at any particular time in the filtrates are the result of a slower
change which has been effected by the nitrifying bacteria upon ammonia
previously absorbed in some physical manner upon the surface of the filtering
material. The procedure for the purification of sewage used in “contact
beds” has been held to assist successively the processes of adsorption of
ammonia and nitrification. Dunbar and Thumm* consider that, in the
“filling” and “full” stages, putrescible and oxidisable substances are
retained upon the surface of the filtering material, and are subsequently
oxidised at times when the bed is full of air, the oxidation being the work of
bacteria in the bed, among which the nitrifying bacteria rank high in order
of importance.
As regards the complex oxidisable putrescible substances of high molecular
weight, the solid suspended matter will be retained, of course, by mechanical
filtration, while the soluble constituents may, doubtless, be supposed to
undergo some physical adsorption.ti But the greater part of the nitrogen
present in sewage is there in the form of free and saline ammonia, and these
are the compounds most markedly retained as the sewage passes through
the filter; yet for such simple compounds as these, adsorption by solids has
been shown to take place only to a small degree or not at all§ To attempt
to explain removal of ammonia by adsorption then, would appear inadequate.
Special experiments were therefore made to investigate the behaviour of
filtering materials with ammonia and its salts ; also during the investigations
in Section I, careful note was also made of any facts which should tend
to confirm or refute the theory of nitrification quoted above.
If the theory of a previous ammonia absorption and a subsequent oxidation
were true, then contact beds should be much more efficient nitrifiers than
continuous filters, but the contrary proved to be the case, the latter doubtless
owing their greater efficiency to their more perfect aération. Again, while
complicated organic substances appeared to be absorbed in the top layer of
the filter (Table III, 3), the disappearance of free and saline ammonia was
shown usually to take place lower down in the filter(Table IIL, 1), and to be
always associated with the appearance of oxidised nitrogen (Table III, 2, etc.).
Moreover, during the maturing of the filters, before oxidation of nitrogen
had occurred, no absorption of ammonia could be detected, although this was
* Dunbar and Thumm, ‘ Beit. zur Abwasserreinigungsfrage,’ 1902.
j Soyka, ‘ Archiv f. Hygiene,’ vol. 2, 1884.
t Kattein and Liibbert, ‘Gesundheitsingenieur,’ vol. 25, 1903.
§ Weppen, ‘Ann. d. Chem. u. Pharm.,’ vol. 55, 1845, and A. Mayer, ‘Lehrbuch d
Agrikulturchemie,’ 1871.
256 Dr. H. Chick. The Process of Nitrification [Apr. 1,
frequently looked for. It was possible, however, that, at the very first,
ammonia had been taken up by the filtering material, to saturation point, and
that afterwards no more absorption was possible until nitrification had
begun. Unfortunately no analyses were made at the very beginning, but
this gap was afterwards filled by special experiments with clean sterile
coke.
The tendency of these observations was thus in opposition to any theory
of ammonia-absorption by the filtering material, and this opposition was con-
firmed by the following experiments, made to test the power of various solids
to absorb ammonium salts. The solids employed were barium sulphate,
sand, and ground-up “clinker,’ and the experiments were carried out as
follows :—
A small quantity of a solid (1 gramme or 2 grammes), previously carefully purified,
was weighed out into a small flask, which was then exhausted to remove air films, which
might cause imperfect contact of solid and liquid.* A measured quantity (50 c.c. to
100 c.c.) of ammonium chloride solution was added through a tap-funnel, and the
whole left standing for 24 hours. The clear liquid was then drawn off by a pipette,
and the ammonia estimated in a small portion (1 to 5 ¢.c.); this was first diluted to
about 500 ¢c.c. with NH,-free water, and then distilled and nesslerised. The remaining
liquid was well shaken up and the muddy residue analysed similarly : a correction had to
be made for the volume of the solid, which was measured, after centrifugalisation, in a
graduated tube. In every case, as a control, a blank experiment was also made, similar
in every detail except that NH,-free water replaced the ammonium chloride solution.
The solids had previously undergone a careful purification by washing, and often, by
ignition also. The ammonium chloride solutions were exceedingly dilute, so as to approxi-
mate to the concentration of ammonia in ordinary sewage.
In Experiments 1 to 3 (Table [X) the ammonia yielded, both by the clear
and the muddy portions of the liquid, was found to have diminished. It was
therefore supposed that boiling was insufficient to drive off any ammonia
which might have been absorbed by the solid; accordingly, in Experiments 4
to 7, a small amount (10 cc. N/1 KOH) of alkali was added before distil-
lation. In this case the analysis of the muddy portion of the liquid showed
a small amount of the ammonium salt to have been absorbed by the solid,
but nothing comparable to the effect required in a sewage filter. Moreover,
the slight removal of ammonia demonstrated would appear to be a chemical
rather than a physical phenomenon, alkali being necessary to free the
absorbed ammonia from the solid.
In none of these experiments, however, was coke itself employed, and the
surface of solid was very small in comparison with the amount of liquid taken ;
* In Experiments 6 and 7 (Table IX) the flask was not thus exhausted, and the agree-
ment of their results with those of previous experiments indicates that this precaution is
unnecessary.
1905. | with reference to the Purification of Sewage. 257
the following further experiments were therefore made, and confirmed the
preceding ones.
Experiment 8.—Exactly the same coke as had been used for the filters was taken and
thoroughly washed and dried. An amount occupying a volume of 30 c.c. was placed in a
flask with 50 c.c. NH,Cl solution of concentration equal to about 5 parts ammonia
per 100,000. In a control flask 50 ¢.c. of the liquid was placed alone. After 24 hours
and after 48 hours the liquids in the two cases were examined, a small quantity (0°5 c.c.
to 1 ¢.c.) being removed, diluted to 50 c.c. with NH;-free water and tested with Nessler’s
reagent. In no case was the reaction fainter where the liquid had been in contact with
the coke. (It was shown that the coke did not of itself yield ammonia by a control
experiment in which NH;-free water replaced the NH,Cl solution.)
Experiment 9.—Coke, which had been thoroughly washed, dried, and sterilised, was
placed in a cylinder to form a small filter, and a solution of NH,Cl (10 parts NH,
per 100,000) allowed to drop slowly through.* The filter occupied a volume of about
1 litre and during the first hour 50 c.c. came through, while in 15 hours a total of 500 c.c.
was filtered. The first filtrate of 50 c.c. was tested for ammonia and compared with the
original liquid. The tint given by the filtrate (after suitable dilution and addition
of Nessler’s reagent) was, if at all, only a shade paler, indicating only a negligible
difference. After a second hour the filtrate was again compared with the control, and a
similar result was obtained. The filtrate coming through in the next 13 hours was
similarly tested, but no absorption of ammonia was detected.t
It may be objected that experiments with raw, cleansed, filtering material
are not applicable to the occurrences in the mature filter, where the surface
of the coke is probably coated, in some manner not yet investigated, and
might possess the faculty of absorbing ammonia in a manner similar to that
already demonstrated in the case of certain colloidal substances.{ Therefore
it is hoped, in the future, to make experiments with matured coke, elimi-
nating, if possible, the action of bacteria. The available evidence is, however,
opposed te such absorption, for it is in the uppermost layers of the filter
that such a coating would be greatest, and yet disappearance of ammonia, at
any rate during the maturing period, has been shown to take place lower
down, and in any case coinciding, both as regards time and place, with
nitrification.
Upon consideration of the experimental data at present available, one is
therefore inclined to reject the current theory of nitrification and to consider
* The concentration of ammonia was greater than in the preceding experiments, where
it approximated to that in ordinary sewage; here a more concentrated liquid was
employed, in order to be comparable with the diluted urine which was then being treated
on the filters.
+ These last two experiments are in perfect accord with some of A. Mayer (‘ Lehrbuch
d. Agrikulturchem.,’ 1871), who showed that pure carbon in a porous condition was
unable to effect any significant absorption with many salts Jong known to be absorbed by
the soil.
t Van Bemmeden, ‘ Landw. Versuchsst.,’ vol. 35, 1888, ‘ Zeitschr. f. physikal. Chem.,
vol. 18.
258 Dr. H. Chick. The Process of Nitrification [Apr. 1,
the disappearance and oxidation of the ammonia to be parts of one process,
which is carried out by the nitrifying bacteria in the time taken by the sewage
to pass through the filters.
For these experimental filters the time taken for the passage was measured
directly.* For the Vienna continuous filters on July 1, 1901, the time was
approximately 34 hours for the tall and medium filters and only 5 minutes
for the short filter (nitrogen oxidation in this filter had not then progressed
beyond formation of nitrites). For the Munich continuous filters on March 17,
1903, the time was 2 hours for the tall filter, and $ hour for the short one.
At this date, a too concentrated sewage was being employed, and the filters
were not at their best, but still four-fifths of the ammonia in the sewage was
oxidised while passing through the tall filter.
Section LV.—General Conclusions.
1. Nitrification of ammonia during sewage purification occurs in two
stages which may be referred to the activity of two classes of bacteria, one
producing nitrites, and the second oxidising the nitrites to nitrates. These
bacteria exist not only in the substance of the filter, but are also carried
away in large quantities in the filtrates.
2. These organisms belong to the same group as those concerned with
nitrification in the soil, isolated by Winogradsky. It is, at first, difficult to
understand how organisms so susceptible to the presence of organic matter
are able to live and do their work in sewage filters. The following, one or
all, form possible explanations.
(a) The nitrifying bacteria may be, to a certain extent, protected by the
presence of other organisms, and this view is strengthened by the results of
certain experiments with the nitrate-producer; in symbiosis with such
organisms, made in the course of the present investigation.
(6) It has been shown that porous materials, such as coke, are able to
retain upon their surface complicated organic substances of high molecular
weight, when these are presented in solution. We may suppose this
absorption (together with the mechanical separation of the suspended
* 100 cc. of a 2-per-cent. solution of sodium chloride were sprinkled over the top of the —
filter (that being the volume of liquid usually delivered at each discharge of the siphon).
The filtrates were then continuously tested with silver nitrate until a copious precipitation
was obtained; the ordinary sewage filtrate yielded only a slight reaction with silver
nitrate. The passage of liquids through such filters is a very complicated process and one
not yet thoroughly investigated, and though, doubtless, the times thus determined may be
considered to apply to the majority of the liquid going on at any particular time, they
must still, strictly speaking, be regarded as approximate and indeed minimal values.
1905. | with reference to the Purification of Sewage. 259
materials in sewage, also largely of organic origin) to take place principally
in the upper layers of the filter. The nitrifying organisms will then be able
to live and multiply lower down in the filter where the amount of organic
matter present will be comparatively small, and this view has been experi-
mentally confirmed in the present work.
(c) It has been lately shown by Wimmer,* in the case of the nitrate
organism, that a porous medium has a markedly mitigating effect when
organic matter is present, and the coke and other materials of which sewage
filters are made, are selected mainly on account of their porosity. It is only
fair, however, to state that Wimmer’s experiments were not made with
absolutely pure cultures, and part of the beneficial effect observed may have
been due to a symbiosis, though, from the nature of his experiments, it
would seem unlikely.
(d) The nitrifying bacteria are doubtless present in very great numbers in
the filters, and this may assist them in withstanding the effect of organic
matter. This view is based upon certain observations of Winogradsky and
Omeliansky,* in which nitrifying organisms, if present in sufficient quantity,
were shown to withstand amounts of organic matter otherwise inhibiting
them.
3. In the maturing of sewage filters, the two stages of nitrification may be
markedly separate in time (Vienna experiments), or may be both developed
together (Munich experiments). This difference is correlated with the
greater or less ammoniacal content of the sewage. In the stronger sewage
used for the Vienna filters, the well known inhibitory action of abundance of
ammoniacal compounds (especially of free ammonia and carbonate of ammonia,
which are so largely represented in the sewage), presumably retarded the
development of the nitrate-producer, until the nitrite-producer was sufticiently
well established to be converting most of the ammonia into nitrites.
4. As a result of special experiments with coke, and of analyses of the
filtrates at different depths of the filters, and at different stages during the
maturing period, it would appear that there is no evidence of absorption of
free and saline ammonia without contemporaneous nitrification. Further
research is necessary, but the theory of a previous physical “adsorption ” of
ammonia and subsequent slower nitrification would appear, at present, to be
without experimental foundation.
5. One is therefore inclined, in the present state of our knowledge, to
consider the process of nitrification, during the filtration of sewage through
* See footnote, p. 242.
+ Lohnis, ‘Centralbl. f. Bakt.” 2 Abt., 13, 1904, and Boulanger and Massol, ‘Comptes
Rendus, vol. 140, 1905.
260 Dr. H. Chick. The Process of Nitrification [Apr. 1,
such filters, to be an extremely rapid biological process, requiring for its
completion only the time taken for the liquid to pass through the filter
(approximately 2 to 3 hours, possibly a little more). The rapidity of the
process is probably to be explained by the very great number of nitrifying
bacteria present and the very efficient aération which obtains. In such
filters also, the general conditions are ideal for quick action, as the continuous
trickle secures rapidity of diffusion, and forms a great contrast to the much
slower effect in stationary fluids.
6. Temperature has a marked influence upon the oxidation of sewage, a
higher temperature being noticeably more favourable. This indicates that
the efficiency of sewage filters in practice would be much increased if at a
reasonable cost they could be artificially maintained at a warm temperature
during the winter.*
7. The previous conclusions are chiefly drawn from experiments with
* continuous filters, but filters working as contact beds were also investigated
and the two methods compared. On the “ammonia adsorption theory,” the
contact method should have proved the most efficient. This, however, was
not found to be the case. The advantages of the continuous method would
seem to lie in the much more complete aération and efficient diffusion, and
also in the stratified distribution in the filter of the different stages of the
sewage purification. Some of the present experiments were quite comparable
with practical installations as regards quantity of liquid treated, con-
centration of nitrogen, etc., and the results were much more satisfactory than
those usually obtained in practice. The obvious difficulty in practical
employment of continuous filters is with regard to the solids in suspension,
which can only be permitted upon the filter to a small extent without risk of
clogging. The present experiments were all made with roughly filtered
solutions, but the difficulty could be met in practice by a previous screening
of the sewage or by passing it through a septic tank. Should clogging occur,
it will probably take place in the superficial layers and could be remedied by
simple mechanical treatment. In the case of contact beds, however, clogging
necessitates the cleansing of the whole bed, an exceedingly costly process.
From these considerations, and as a result of the present experimental study,
the method of continuous filtration would appear to be a most advantageous
method of purifying sewage.
* Ducat filters, which are artificially warmed in cold weather, perform an amount of
nitrification which is well above the average.
1905.]
per 100,000 by weight.
Tables I—IX.
In these tables the estimations of “free and saline ammonia” are expressed as
ammoniacal nitrogen, those of “albuminoid ammonia” as albuminoid nitrogen, those of
nitrites as nztrous nitrogen, and those of nitrates as nitric nitrogen, all in parts of nitrogen
with reference to the Purification of Sewage.
261
Nitrites were estimated by the metaphenylenediamine colorimetric method unless
otherwise stated, the sum of nitrites and nitrates by the indigo method (Vienna analyses),
or by the copper-zinc couple method (Munich analyses).
Throughout these tables the grades of reactions are represented as follows: reaction
absent by 0, a trace, or faint reaction, by f, definite reaction by +, and intense reaction
by ++.
Oxidisability is expressed in parts oxygen absorbed per 100,000 by weight, less that
In figures marked with an asterisk the oxidisability
absorbed by any nitrites present.
was approximately gauged by the difference between the oxygen absorbed in the cold and
on boiling.
Table Is.—Vienna Continuous Filters, showing the Progress of Nitrogen
Oxidation during Maturing. Filters all started on February 20, 1901.
Figures in brackets are weeks elapsed since the start.
Course of oxidation. Tall filter. Medium filter. Short filter.
Oxidised N. first detected in| March 18 March 21 March 28
filtrate (4 w.) (4 w.) (5 w.)
Nitrites present in quantity, no March 28* March 28+ July 1f
nitrates (5 w.) (5 w.) (19 w.)
Nitrates also present in quantity May 24 May 13
(11 w.) (94% w.)
Nitrates alone present ............ July 1 July 1
| (19 w.) (19 w.)
* 10 parts per 100,000 N. as N.O3.
+ 13 parts ditto.
{ 4:1 parts‘ditto, nitrates also present in small amount.
Table Is.—Munich Experiments. Filters all started April 1, 1903.
Continuous filters. | Contact filters.
Course of oxidation.
Tall. | Medium. | Short. Tall. Medium. | Short.
| |
Oxidised N. first de-| May2 | May 2 May 2 May 16 May 16 May 16 |
tected in filtrate (4 w.) (4 w.) (4 w.) (63 w.) (63 w.) (6% w.)
Nitrites present in| May 18 May 23
quantity, nitrates | (7 w.) (7% w.)
present
Majority of oxidised N.| May 27 — — | June 25 | June 25 | June 25
present as nitrates (8 w.) ~ (12 w.) (12 w.) (12 w.)
Nitrates alone present.... June 8 June 13 | June 13 | July 9 July 9 July 9
| (0 w.) (103 w.) | (103 w.) | (143 w.) | (14h w.) | (142 w.)
[Apr. 1,
The Process of Nitrification
Dr. H. Chick.
262
= 8-8 = T-8 0 41-0 30: 0 ceeeeees | Tye9—oq@dgT | 8O'L'ST 98
= I: 61 = 0) 0 L- 0- OT a 'd [fourm smo | ¢0'/'eT 9g
19. T g. 8 = £.8 0) SI-0 80: 0 "| TBI —oqeryL | g0'9'FS rE
I OT 0: LT = 0 0 8-8 3: ST “9 'd T ‘our smo | ¢0'9%% 6
L-P 0-% = LT ) ¥- O 10-0 “" umipea— “| eg
9-8 8.3 = I. ) 2-0 (AON) asa Te 99849, heoguur-st 1g
6: OL 9. — (0) (0) 9.T ONee = aes “ oSeMos MBIT oe
= 6-2 = 0-3 9-0 Z-0 -0 pee 11®}3— 998191], ua 6
aoe oe = i ‘ ae saa eee ned. GRUMG! den teo G' 12-98 { cs
os 0. == G. 1 Fé TL z-0 1-0 unIpsu—ajyeuytT | OSes | LB
= 0. a ) 0) ¥T 9-8 aoa oBUMOS MUY —-O'S"EZ 9
ard g.8 = ¥-0 G.% 3-0 ClOy ge i Sas 1%" S0'SET | Ge
Q. Il I. G as, oO 0 Q. I Cc. @ seen eeeeenes asemos MOY 20'S'6T ind
= = mr f LP = ERG gee PEE S10U8 ee LOWS £3
sige = = 0-g f = LF “ umipem— “| TOL'T ra
= = = 8. L 0 = DOs Nae T133— 9984 = LOL 1z
ae es ne 0 0 ae 6- TI eee eeeceseee aS'eMos MBIT | TO“ 0z
= = = LT 0-9 = SHO see koes 13— =“ TO" "FZ 61
3G I-81 = = if 8-1 SHOR <1 |e qtoys— 81
«1-F g- 6 = L-& L- F-0 €-0 “+ UNIpeni—oqecy [Ly \o'g'st-et LT
Gc. ST BLL —_— (0) (0) Gg. ps 7 i ae OCDORGROD esemos Muy | 91
= G- OT = i) ¥- OL €-8 Sagres T®3—9F244LT | LOFT ST
fame lL. 91 4 LI 0) 0 0: Zz fap FL een eeeerence aseMos MOY | 10'r'T PL
9-F = = = f = PeGiley 2 eee qtoys— “| 10°88 81
= = = 0) 9-1 = T- ST “* umipeu— “ | T0's'sz ‘ae
T-¢ 9: IT = 0 I- OT 88. 0 Cole liane * T1@3—e4TLT 10°88 II
8: BP @- LT 3: AT ) 0 0:3 CoG we. See esemes MeY — 10'S'8Z OL
¥-G = = 0 0 = aie Sioa qtoys—ozeryLy 10'8ST 6
&. ST — — 0 0 = Pan leeeaearee: osumes Muy 10'S ST 8
9. 9 noes a 0 (0) = — | ~—_«jodaacop56 910 S— “cc 10'@’eL L
3: = = 0 ) = aay oe Woe TI} —998t4LT | TO'S'ST 9
e. CL eis aes (0) 0 nee: —~ —— “(| J0000G000 eee osemos Mey | 10'2'E1 G
Pp. 9 Book = 0 0 a 2 me (ee 410 s— “ 1O'e'L ,
p- = = 0 0 = aaa J|° wmtpem— “ TO's'L g
6.¢ = = 0 0 = Cae Sees TIe3— eT | TO'eL z
©. OL aes = 0 0 Le DFA Jews n ee eeeeee aSemos MBI Toe" I
‘© Jo sunI0e4 ur *poye[noeo Tqeppely “ueS01410 ‘ues01}1U “uoSdo1q1U “u830141U “oq JO FUSIOP oy “ssf BUY
‘AqTIqesIpIxQ | Uesomy1U [eyo], | ‘Mesoaqra [ey07, OLAGINT SNLOT4I NT plourmmaly | [vovmomumy | “poskyeue [eLeyepy a JO ‘ON
000°00T 10d sqred
UI pesserdxe oi sy[NseL OT, “EOP T poyeqs [[e ‘sieq[y Snonuryuoo yoruny oY} YIM [eop 9¢—Fz sesdjeuy {10'Z'0Z%
pojtegs [[e S109} SNonUTZUOD vUUOIA EY} YIM Teop SZ—T sesd[euy ‘soqvIq[IA pue JUON_V esemog jo sosd[euy— J] 9]qeI,
1905. | with reference to the Purification of Sewage. 263
Table I1I.—Showing the Course of Oxidation at Successive Depths in
Tall and Medium Filters (Vienna).
| he Filtrate at successive depths. |
|
[= = —
No. Date. Constituents. | Sewage.
Tap No.1, Tap No.2, Tap No.3, | Tap No. 4,
| | 50cm. 100 cm. 150 cm. | 200 cm.
| ‘Pall filter. | |
1 24.5.01 | Ammoniacal N. eel 278) POF 2°27 0°39 0-29
2 24.5.01 | Oxidised N....... Vai XO ah + ++ +++
3 27.5.01 | Oxidisability Oe) i oad 2 -6* SEOMS |) 2e38*
4 27.5.01 | Nitrites............ _ ye St No. 2x5 | No.2x7
5 1.7.01 | Ammoniacal N.} 11°9 2°5 0°2 0 -04 0°06 |
6 1.7.01 | Nitrites............ 0 ofa 0 0 0
7 1.7.01 | Nitrates ......... 0 = + + 7°8
| Medium filter.
8 | 17.01 |Ammoniacal N.| 11:9 | 81 | 4°12
9 | 1.7.01 | Nitrites............ | 0) if fe
10 | 17.01 | Nitrates ......... Liat ¥. 4°96 |
In 2 the test was made with diphenylamine, in 3 the estimations are approximate.
The high numbers in filtrate of 8 are due to deterioration in efficiency of the filter following
partial clogging.
+ Nitrous N = 6:0, nitric N = 111.
Table 1V.—Munich Continuous Filters from June 24, 1903, to November 27,
1903, using Cows’ Urine diluted 1 in 100; this contained 14—17 parts
NHz per 100,000 and no oxidised nitrogen. In cases marked with an
asterisk the urine was only diluted to 1 in 50.
| Tall filter. Medium filter. | Short filter.
|
Slate ES) Nitrite!) Nitrate ee | Nitrite | Nitrate) NH, | Nitrite Nitrate
per re- re- werere= "||P Sore. | er re- re-
100,000. | action. | action. 100,000. | action. | action. |100,000.| action. | action. |
| |
| | | | |
26.6.03 | | 0 ++ je (6) } ++ | 1 ifs ++
| 30.6.03| O72 | O ++ Ol O | ++ 2—3 fa ++
9.7.03} 0-05 | Oe a Ee. | 2—3* + | +t 1—2 + ++ |
13.7.08 | | | } | }
aa | +0-2 | Doe sear | eae SF POV NN joerc Sg | (ee fess |.
| 23.7.03| 0:05 | O | ++ | 4—5* Sy renee (eee Ae 1—2 + ++ |
| 24.10.03 | 0°05 0 ++ 0-05 0 | ++ On| 0) Pee
_ 27.11.03 | 0-02 10) ++ 0-04 0 GEE 0-04 0 ++
| 20.104) 5:0* | Ff ++ |8—10¥| ++ | +4 15* + ++
The ammonia estimations by direct Nesslerisation are only approximate ;
or inconsi
derable).
nitrites were tested
for by acidified starch-zinc-iodide; nitrates by diphenylamine (only specific if nitrites are absent
264 Dr. H. Chick. The Process of Nitrification . [Apr. 1,
Table V.—Enumeration of Organisms in Filtrates.
No. of organisms per cubic centimetre.
F Time sub-
Date: 2) Mupiehicon: cultures were
2 Growing in Nitrite- Nitrate- Kept.
bouillon. producer. producer.
6.6.03 Mall: Ce etosnencsescrs 10,000 10,000 100* 6 weeks
6.6.03 Medium ......... 1,000 10,000 100 Co,
6.6.03 | Short.........000. 100,000 10,000 100 65
12.6.03 Nl lee Sere eenonancr 10,000 10,000 10,000 4, months
12.6.03 Medium ......... 100,000 100,000 1,000 4
12.6.03 | Short...........00 100,000 10,000 100 ee ae
NS1G.O37 4 Dall eranacccesteee 10,000 10,000 100 Former 4 m.
TSIG!03) Ml Shortie cere 1,000 1,000 10 { Latter 6 w.
24.6.03 Stalls Goaenosaveooene 10,000 1,000 10,000 4, months
24.6.03 Medium ......... 10,000 1,000,000 10,000 yas
27603) | iiglla eee etecee: 1,000 = 10,000 A
27.6.03 | Medium ......... 10,000 = 10,000 is,
* Culture containing 0°01 c.c. filtrate, used for dilution experiment on p. 252.
Table VI.—Results of Inoculating two different Impure Nitrite-producer
Cultures, “a” and “db,” into Ammoniacal Media, both directly and after
passing through one or two generations in bouillon. ++ indicates an
intense nitrite reaction.
3 2 5 “a,” three | “a,” twelve | “6,” three “6.” four
SPeGs.|) Seibenw ar sneer oe. weeks old. days old. weeks old. | weeks old.
i From original material ...... ++ = = ++] ++ apr
A From Series 1 ...............05 — Fe | bt Pa | ae =
3 From original through bouil- 0 @) 0 ++] ++ ++
lon (Series 2)
6 From Series 2 again, through — _— = (0) 0) _
bouillon (Series 5)
1905. ]
with reference to the Purification of Sewage.
265
Table VII.—Results of Inoculating into Nitrite Medium of Five Strains
of a “Symbiotic” Culture of Nitrate-Producer, “d,” after Successive
Generations in Bouillon. + indicates a positive, + + an intense reaction.}
ce d 1 ” (77 d 2 ” | “cc d 3 ” ii3 d 4? | “ce d 5 ”
Dates of Date of Date of
successive | inoculation aoe g |
inoculation from bouillon eerie ale eo ee ee ie eee
into into nitrite | Preceding | $/8/28)/2/2/28|/2)/2|28)3
bouillon. medium. cultures: lee Boe rer |B |e | 8 | & £
|
17.12.03 -» 23.12.03 12.1.04 O f+ +! O }++ ++
12.104 + 18.1.04 3.2.04 oy) | 0 leans O |++|) O j++} O |+4
18.1.04 > 26.1.04 10.3.04 ++)| 0 (0) aaa O |}++| O |++/+-+4) 0
26.1.04 > 3.2.04 HOBO, Wes | Ogee! OP ee ca
Table VIII.—Showing the Different Behaviour of Pure and of “Symbiotic”
Bouillon-Cultures of the Nitrate-Producer when Inoculated into
Nitrite Medium, the latter tested for nitrites and nitrates 31 days
after inoculation ; + indicates a definite, + + an intense reaction.}
: ‘Symbiotic ” cultures, bouillon
Renu One oF Pure cultures, bouillon clear. showing growth,
the nitrite as
medium. | |
No. 25. No. 39. No. 67. No. 49.| No. 29. | No. 12.| No. 21.
Nitrite ...... adr || shar |] apap || apap || apap || ab oe 0 OF) 0 0 ee be
Nitrate ...... 0 0 0 (0) (0) 0 ++ ieee ++) ++ + | +
Cultures No. 49 and No. 29 were strains of culture “d’’; cultures No. 12 and No. 21 were of
different origin.
{ With cultures still containing nitrites, evaporation with ammonium chloride preceded the
diphenylamine test for nitrates.
266 Nutrification with reference to the Purification of Sewage.
Table [X.—Experiments upon the Absorption of Ammonium Chloride
from Dilute Solution by Various Finely Divided Solids.
Original solution of
NH,Cl added.
Liquid after standing,
ammonia content per
Ammonia in
blank control
in thousandths
UL OSEGD joists. of a milligramme.
No Solid tested ;
weight taken. hone
Volume content Upper Lower :
of liquid per clear turbid i. ae foe
in c.c. | 100,000 liquid. liquid. ques |) saute
parts.
1 BaSO,, 1 gramme 100 1:00 1°02 0°91 0 0)
2 | Sand, 2 grammes ...... 100 5 ‘00 4°30 4-04 2 0
3 | Sand, 2 Pe eee 50 5 00 4-14 3°78 3 (0)
4 | Clinker, 2 grammes ... 50 1-00 0°82 0°89 2 0
5 | Sand, 2 grammes ...... 50 1-00 0°75 1-03 5 0
6 | Sand, 2 san eliiys abiis 50 1-00 0°83 1°05 3 (0)
Th | ISEMICY cco cassedescnsescne-0 — 1-00 0°85 0:99
In Experiments 4—7, 10 c.c. N/1 KHO was added before distilling off the ammonia.
267
The Action of Anesthetics on Inving Tissues. Part 1—The
Action on Isolated Nerve.
By N. H. Aucock, M.D.
(From the Physiological Laboratories of the University of London and St. Mary’s
Hospital Medical School.)
(Communicated by A. D. Waller, F.R.S. Received November 9,—
Read December 14, 1905.)
CONTENTS.
PAGE
Ti, UiatirroehCBKOr ..ocoodcoodaoadonnocongonhangadoeceodso0or BeoasedaCobangoD wugeoudanbasodupan60000 267
II. Experiments.—Series I. Anesthetic to Whole Nerve.................00ceeeeees 267
Ill. Series I1.—CHCl, to Parts of Nerve in varying Percentages ............... 269
IV. Series I1I.—Fixed Percentages, CHC], and Ether ................:scseeeeeeeees 271
V. Series [V.—Simultaneous Anesthetic “ Balance” Experiments ............ 273
VI. Series V.—Electrical Resistance of Nerve after CHCl,, and Control
IX PELUME NUS prreccceeecee eee hce mee sos etesch once ser cleececenaeccesscaelsaceacle 275
VII. Series VI.—Electrical Resistance of Nerve after Ether ...............:000.0008 278
Wa lelibie Observations iivnssssachschenasscesse ce satins cecuesaecuasseasei odie avecevassueraaaneseens 280
Introduction.
The action of anesthetics on isolated nerve has already been studied by
Waller* as regards the effect on the negative variation in the sciatic of the
frog, and by myself in mammalian nerves.t In the course of these
researches it became evident that the anesthetics used (chloroform, ether,
carbon dioxide) affected not only the negative variation, but also the injury
current, and as this action has not been studied before, as far as I am
aware, it seemed desirable to investigate the matter not only in nerve, but
also in other tissues.
' The inquiry falls naturally under two heads: first, in how far the
phenomena throw light on the processes of nerve action ; and, secondly, as
regards the chemical and physical action of anesthetics on the animal
protoplasm generally. These are obviously only parts of the same story,
but for convenience I have considered the subject mainly under the first
heading in this part, leaving the more general question to a future occasion.
Experiments.
If the sciatic nerve of a frog be taken and a fresh transverse section be
made at the distal end, and this end be placed in contact with one non-
* Waller, “Lectures on Animal Electricity,” 1897, and ‘Proc. Physiol. Soc.,’
November 13, 1897, ete.
t ‘ Roy. Soc. Proe.,’ vol. 71, p. 264, and vol, 78, p. 166.
VOL. LXXVII.—B. U
268 Dr. N. H. Alcock. [ Nov. 9,
polarisable electrode, while another electrode rests on an uninjured
longitudinal surface, the galvanometer will indicate a difference of potential
which declines at a certain rate. This can be measured either by photo-
graphing the movement of the galvanometer spot, or, more accurately, by
balancing against a known potential, reading the potentiometer at convenient
intervals of time, and plotting the figures on squared paper; this latter
method eliminates any change in the resistance of the object. The curve
obtained is concave to the abscisse, neglecting a variation which is occasionally
found during the first five minutes of the experiment.
If, when this curve has assumed a typical form, CHCl; vapour be applied
to the nerve, a sudden drop is observed. If the vapour is weak, this is
followed by a recovery when the CHCl; is removed, if the vapour is strong
(12 per cent. or over) the drop is permanent (fig. 1). This may be due
Li
-
200 Fs
190 f Ee
180
-
170} (106)
160
Fie. 1.—Sciatic of Frog. CHCl, vapour at 12°5 per cent. between the vertical bars.
Ordinates 1 mm. = 00001 volt. Abscissee 1 mm. = 15 seconds.
1905. | The Action of Anesthetics on Living Tissues. 269
to events occurring either at the cut end, or at the longitudinal surface or
both, the experiments in Series IJ were undertaken to determine which of
these factors predominated.
Series LT.
The nerve-chamber was divided by a transverse gas-tight partition of
modelling wax, so that the aneesthetic could be applied either to the cut end A
or to the longitudinal surface B (fig. 2). The figures obtained from the
To sec. coil Y, d
Y
Fic. 2,—Nerve chamber.
potentiometer readings have been plotted out to a scale on the ordinate
of which one division = 0°0001 volt, and on the abscissa one division
= 15 seconds. The point at which the anesthetic was applied was taken as
the zero, and the relative heights plotted from this, the values are, therefore,
relative and not absolute (fig. 3). The figures under the middle part of
each curve indicate the percentage of CHCl; vapour, as measured by Waller’s
densimetric method.* The vapour was contained in a bag of gold-beaters’
skin; the leakage from this was ascertained to be small during the time
taken by an experiment of this kind. Here, as elsewhere in this paper,
I have only considered experiments where the results are sufficiently
concordant to admit of a consecutive series being employed without having
to reject isolated experiments. Any unsuspected errors are, therefore, of a
constant magnitude throughout a given series.
It will be seen that—
(1) CHCl, to cut end of nerve causes an increase in the injury current.
(2) This increase is roughly proportional to the strength of the CHCl;
vapour up to about 12 per cent., greater concentration then gives no further
increase.
(3) CHCl, to longitudinal surface causes a decrease of the injury current.
* Waller and Geets, ‘ British Medical Journal, June 20, 1903.
U 2
Exp.926
G90)
+- EXp.929
pit \ao%
fe)
©
Oo
6
iS
Exp.927
(126%)
fo)
fe)
“I
Q ©
i fe) O
(o)) Ov
Q
ce)
©
— Exp.930(17-9%
Tes 5 etc. minutes.
Fig. 3.—Series II. Relative increase and decrease of injury current by CHCl, vapour at different strengths.
Ordinates 1 mm. = 0:0001 volt. Abscisse 1 mm. = 15 seconds.
-OLO
The Action of Anesthetics on Living Tissues. 271
(4) Recovery takes place when the vapour is weak (9 per cent. or less),
not occurring when the strength is 12 per cent. or more.
Series ITI.
The method adopted in Series II, while suitable for percentages of CHCl
from 1 to 10, is defective in many ways, and an apparatus was designed to
deliver a constant high percentage of CHCls, saturated with water vapour, and
at a constant temperature. This consisted of a foot bellows with a branched
delivery tube, one limb of which passes to seven Fresenius flasks, the first
three containing CHCl; the next water, the next two contain a mixture of
CHCl; and water, and the last is empty. The apparatus delivers moist
CHCl; vapour at a concentration of 13 to 17 per cent. (depending on the
temperature of the first flask) with a variation in strength of about
0:5 per cent. and a temperature variation of about 04° C., and by estimating
the strength of the vapour after it has passed through the nerve-chamber,
and noting the temperature before and after each experiment, these residual
errors can be allowed for. The five flasks on the other limb of the air
supply contain water, and a small stream of air passing through them
serves to ventilate the other part of the nerve-chamber, thus guarding
against accidental escape of CHCl;. A similar apparatus is used for ether:
in this case the percentage is about 45. Unless otherwise stated, all further
experiments were made with this apparatus, with occasional modifications.
In this series the strength of the CHCl; vapour was 12°5 (-0°5 per cent.),
and was applied for 10 minutes. The details of the various experiments
differed. The values are now absolute, and have been corrected for electrode
eurrent. Each ordinate division = 0:002 volt; each abscissa division
= 15 seconds.
In Experiments 108 and 107 (nerves of Frog A) the CHCl; was applied
first to the longitudinal surface and then to the cut end. In Experiment 109
(Frog B) the same course was followed, only without making a fresh section,
the curve therefore begins close to the zero line. In these three experiments
the curves fall at first, the effects being naturally opposite in sign to those in
the next three experiments—Nos. 111, 110, and 117—where the cut end is
first affected. No. 110 is practically a repetition of Series I, No. 111 ‘the
same without a fresh section, and No. 117 shows the effect of ether applied
for five minutes instead of ten.
Considering the first half of each experiment—
(1) CHCl (Experiment 111) and ether (Experiment 117) to the distal end
of a nerve cause a rise in potential, approximately equal in amount.
Volt CH Cl; CH CL,
-020\—,—_ #4
I 234 5 minutes
Fig. 4.—Series III. CHCl, of 12°5 per cent. and ether of 50 per cent. to either end of the nerve
alternately. Ordinates 1 mm. = 0°0002 volt, abscisse 1 mm. = 15 seconds. Expts. 107
and 108, CHCl, to L. surface and then cut end. Expt. 110, cut end and then L. surface.
Expts. 109 and 111, CHCl, to different ends without a previous section. Expt. 117, ethers
50 per cent. to cut end and then L. surface.
The Action of Anesthetics on Inving Tissues. 273
(2) The effect of a previous section (110) is to produce a somewhat larger
rise.
(3) CHCl; to the proximal end produces a fall in potential (107, 108, 109).
When there is no previous section (109) the experiment is obviously identical
with Experiment 111; when there is a previous section, the total effect is less.
In other words, if
« = effect of section, and
y effect of CHCl; in the first part of each experiment,
Exps. 109 and 111 = y,
LO hs a7,
ne = 2+Y.
These results are discussed subsequently in connection with the question
as to whether z and y are due to the same mechanism in the nerve or not.
Later experiments (Series V and VI) point to an affirmative answer ; as
regards this present series, it appears that y is of the same order of magnitude
as 2, whether CHCl; or ether is used, and that y has the same direction of
current as w.
A few experiments were made with alcohol vapour; this causes an effect of
the same character as CHCl; and ether, but as it also causes an enormous
increase in the resistance of the tissue, due to drying, the experiments were
not proceeded with further.
When, in the second part of each experiment, the anesthetic is applied to
the other part of the nerve, the final result is similar to that of Series I,
where the whole nerve is at once anesthetised. As can be seen, there is
a small residual E.M.F., occasionally (in other experiments not quoted here);
this may reach a value of 0:005 to 0:006 volt, the exact significance of this
is still uncertain.
Series IV.
In this series, CHCl; and ether vapours of different strength were applied
simultaneously to both sides of the nerve. The result could have been pre-
dicted from an examination of Series I and II, but is a useful control, as
certain sources of error are eliminated.
Experiment. | Central End. Peripheral End. | E.M.F. initial. E.M.F. final.
|
| per cent. per cent.
931 51-0 (ether) 16 ‘1 (CHC1l,) +33 -— 1
932 | 16:1(CHCL) | _ = § —238
933 | 12-5 (CHCl) | 16-5 (CHCI,) +13 =e
a 8:0 (CHCl) | : —184 (a)
o34{ § | 43 (oHeL) jaa 5 (CHCl) | —16 ate (a)
113 | 54-B2°5 (ether) 12-3 (CHCI,) | —25 = 7
274 Dr. N. H. Alcock. [Nov. 9,
If the small final values under 20 be disregarded, these experiments show
that :-—
(1) Ether vapour of 51 to 54 per cent. and CHCl3 of 16 to 12:3 per cent.
are identical in their final effects.
(2) CHCl; of 8 per cent. has a less effect. » CHCls vapour of about
12 per cent. has, therefore, a maximum action.
(3) Ether acts more quickly than CHCl, (fig. 5).
Fic. 5.—Series IV.
Control Experiments.
In order to exclude any possible source of current from electrodes or any
unexpected physical cause, control experiments were made with threads
moistened with (a) salt solution, (>) egg albumen, (c) egg yolk, and (d) fresh
blood. These were treated in an exactly similar manner to the nerves in
Series II. No electromotive phenomena followed the application of CHCl;
vapour. So also nerves kept in salt solution until they gave no injury
current showed no results with anesthetic vapours. HCl and NH,OH gave
electromotive effects when applied either to threads or nerves, HCl with the
same sign aS CHCl;, NH,OH with the reverse. The similarity is therefore
only apparent and probably due to diffusion into the electrodes.
1905.] © The Action of Anesthetics on Inving Tissues. 275
Series V.
The effect on the electrical resistance of the application of anesthetic
vapours is of great importance in endeavouring to give an explanation of
the facts recorded above, and so, although the question has already been
studied by Waller,* I have performed a considerable number of experiments
in order to eliminate as far as possible the numerous sources of error present.
The method finally adopted was to place the nerve on non-polarisable
electrodes in a chamber, pass in air saturated with water vapour, and take
several successive readings until either they remained constant or else the
rate of change was determined. Moist CHCl; vapour was then admitted
for five minutes, a reading taken, the chamber washed out with moist
air for four minutes, and the final value observed. A linen thread wet
with M/10 NaCl solution rested on non-polarisable electrodes in the same
chamber, and readings of its resistance were taken alternately with the
nerve. A thermometer as close to the nerve and thread as possible gave
an approximation to the temperature; as will be seen, the mean variation
of this is about 0°4° C., and in view of the difficulty of securing an equal
amount of moisture and an equality of salt content in the electrodes, threads
and nerves, the temperature error may for the present purpose be disregarded.
Two methods of determining the resistance were used :—
(1) The ordinary Wheatstone bridge, using non-polarisable electrodes and
a constant current from one Leclanché cell.
(2) A slightly modified Kohlrausch apparatus, using the same electrodes
and alternate currents.
With the control threads the results were identical; both sets of readings
show a diminution of resistance of about 2 per cent. after CHCls, in part due
to temperature alterations, in part to other causes. The nerves on the other
hand gave markedly different figures with the two methods.
* Waller, ‘ Proc. Physiol. Soc.,’ November 12, 1898
[ Nov. 9,
Dr. N. H. Alcock.
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278 Dr. N. H. Alcock. [Nov. 9,
These figures show that—
(1) Nerves tested by Wheatstone’s bridge show an apparent diminution of
resistance after CHCl; of about 15 per cent.
(2) Nerves tested by the Kohlrausch method show no alteration of
resistance.
The difference between the two methods is that any polarisation in the
nerve appears by the first as an added resistance. The figures in the first
column of A therefore represent resistance and polarisation, and the
conclusions follow—
A. CHCl; diminishes the polarisation of nerve ;
B. CHCl, does not alter the real resistance of nerve within the limit of
error of the method.
If the diminution in the control experiments is taken as a correction to be
applied to the nerves, the resistance of these after CHCl; is increased by
1 per cent., at present it is doubtful if this correction is to be applied.
-~- [Note added December 12.—A fresh series of experiments (Nos. 211
to 214) gave the final ratio for nerves slightly Jess than the control threads.
The probable limit of error of the resistance experiments in the text is about
+2 per cent., and as this lies in the object examined rather than in the
measurements, greater accuracy seems at present unattainable.]
Series VI.
Similar experiments were made with ether, taking readings by both
methods. The control threads now show a diminution of about 5 per cent.
and an increase during etherisation. Except in this latter particular the
nerves gave results almost identical with CHCl.
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280 Dr. N. H. Alcock. [Nov. 9,
These figures show—
(1) Ether diminishes the polarisation in nerve.
(2) Ether does not alter the real resistance of nerve (with the same reserve
as to the control experiments).
Comparing the result of the two anesthetics, and taking the mean
figures :—
Mean resistance | Mean resistance R./R
before. after. a/ Ry
Nerves CHC wrrcrerecters 121,400 103,500 0 *854 Wheater
se Veber so ae ee: 143,180 122,910 0 865 Care nee
Nerve, CHCl, ............ 122,600 121,200 0-989 :
De eal 75,500 75,000 | 0-994 | Kohlransch.
The final effects of CHCl; and ether are identical, within the limits of error
of the experiments.
Observations.
The full discussion of the problems raised by the facts here recorded must
be deferred until the results are considered of experiments on other tissues
and on the question as to the actual effect of the action of CHCl; and ether
on proteid solutions.* Taken as they stand, the present experiments show :—
(1) That chloroform and ether (and probably alcohol) produce an electro-
motive effect when acting on a frog’s nerve, which has a maximum value of
about 0:030 volt, and the same sign as the current of injury.
(2) That CHCl; and ether produce no alteration of the resistance of the
nerve (within the limits of error), but diminish the polarisation.
Two inferences present themselves—
A. That the electromotive effects are due to the same cause that produces
the injury current.
B. That as the resistance is not diminished no additional ions are formed.
The discussion on the correctness or otherwise of these inferences is post-
poned for the reasons given.
I have again great pleasure in acknowledging the kindness of Dr. Waller,
both for the permission to work in the laboratory of the University of
London, and for advice in the conduct of the experiments. Also to express
my appreciation of the assistance of Dr. B. J. Collingwood, in conjunction
with whom many of the experiments were carried out. Mr. Shapiro has also
given me much help in the later stages of the research.
* See Moore and Roaf, ‘ Roy. Soc. Proc., and also Waller, doc. c7t.
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B.
VOL. LXXVII.
284
Further Work on the Development of the Hepatomonas of Kala-
Azar and Cachexial Fever from Leishman-Donovan Bodies.
By Lreonarp Rogers, M.D., F.R.C.P., F.R.C.S., B.S., Indian Medical Service.
(Communicated by Sir Michael Foster, K.C.B., F.R.S. Received October 16,—
Read December 14, 1905.)
[Puate 7.]
In 1903 Lieutenant-Colonel Leishman, R.A.M.C., described certain bodies in
the spleen of a fatal case of chronic fever in a soldier invalided home from
near Calcutta, which he considered to be degenerate trypanosomes, on
account of their resemblance to the breaking-up dead trypanosomes found in
the spleens of rats 48 hours after death, and he therefore suggested that
trypanosomes might be present during life in this class of fever. Major
Donovan, I.M.S.,(2) of. Madras, however, very shortly after showed that
Leishman’s surmise was not correct, as he found similar bodies to those
described by Leishman in fresh blood obtained by puncturing the spleen
during life, but no trace of trypanosomes; and after examining Donovan’s
specimens, M. Laveran (3) pronounced the parasite to be a Piroplasma, and
suggested the name Piroplasma Donovani for them.
Donovan (4) also claims to have found the parasites in the red corpuscles
of the peripheral blood, but his coloured illustrations of them very closely
resemble ring-parasites of malaria, and have only one chromatine body, and
his statements in this respect have not been confirmed by any other observer.
Ross (5) suggested that the parasite probably belonged to a new genus, and
proposed to call it Leishmania Donovani, and Manson and Nuttall also
favoured the view that it is a distinct genus. In the following year, 1904,
on my return to India from leave, I commenced an investigation of the
subject, with a view to finding further stages of the life history of the
parasite which might throw light on its true nature and classification. In
the meantime Lieutenant Christophers, I.M.S., had been placed on special
duty by the Government of India to investigate the subject, and after
making a careful study of the parasite in different tissues of the body, he
suggested that they might be spores of a microspordium. (6)
My first endeavour was to find some method of keeping the parasite alive
outside the human body, and after a number of trials success was obtained
by preserving spleen blood containing the parasites under sterile conditions
in a cold incubator, preferably at about 22°C. For this purpose the fresh
blood obtained by spleen puncture during life was placed in small sterile
Development of Hepatomonas of Kala-Azar, ete. 285
test-tubes containing a few drops of 2 to 5 per cent. citrate of soda in
normal salt solution, in order to prevent clotting. Under these conditions
not only did the parasites remain alive for many days, but they also
multiplied very rapidly, became much enlarged, and after about three days
some of them developed into elongated flagellated bodies, which I took to
be a stage in the development of a trypanosome, although no undulating
membrane was yet present. This discovery was announced in the ‘ Lancet ’
of July 23rd, 1904, a few uncoloured illustrations were published in
September, (7) and a fuller paper tracing the stages of the development day
by day, with a coloured plate, in November (8) of the same year. Confirma-
tion of my discovery was first furnished by my assistant, Dr. G. C.
Chatterjee,(9) working in my own laboratory, and next independently
by Lieutenant Christophers, (10) working in Madras. Thirdly, Captain
Statham (11) and Lieutenant-Colonel Leishman also obtained the development,
and published their account of it, March, 1905.
During the past year I have made a large number of experiments on the
conditions affecting the development of the parasites outside the human body,
with a view to obtaining a clue to the natural mode of infection, and early
in the present year I published a summary of the results obtained. The
most important conclusions arrived at were, that sterility is essential
to the continued development, and that flagellation takes place much more
uniformly and regularly if the citrated spleen blood is faintly acidified with
citric acid: facts which strongly point to the stomach of some blood sucking
insect as the natural place of development of the parasite outside the body,
and I gave some clinical reasons for considering the common bed bug to be
the most likely conveyer of the disease. So much more abundant develop-
ment of flagellated stages has recently been obtained by the use of acidified
blood medium, that I have been able to make a more satisfactory study of
the exact mode of development, and to come to a definite conclusion
regarding the ultimate stage it reaches, and therefore propose to describe and
illustrate these later stages more fully than in my previous papers, and to
briefly discuss the bearing of the conditions affecting the development of the
flagellated stage of the parasite on the probable mode of infection of the
clisease.
Stages of Development of the Parasites observed in Acidified Citrated Blood.
In the first place, the development in acidified blood is much more
uniform than that obtained by the previous method, so that instead of
finding all stages present after three or four days, with a great preponderance
x 2
286 Dr. L. Rogers. Development of Hepatomonas of [Oct. 16,
of the smaller oval forms, and but few flagellated ones as in alkaline blood,
in the acid medium the great majority of the parasites will be found in
nearly the same stage on any given day, and nearly all become flagellated
after a few days. The sequence of events during the first two days is
the same as I have already described, (8) and they are well shown in the
first two lines of the accompanying plate—all the figures in which have been
drawn to the uniform scale of 1500 diameters magnification, with the aid of
a cainera lucida. Line I shows the parasites seen in a film of spleen blood
made at the time it was obtained, and consequently before any development
had taken place. After incubation for two days at 22° C. the forms shown
in Line II were present, figs. 1 and 2 showing considerable enlargement,
especially of the macronucleus and protoplasm of the body. Figs. 3 and 4
also show the earliest appearance of the eosin-staining body, which is repre-
sented as a clear space in the drawings, but is of a rosy-pink colour
with Romanosky’s stain, and quite distinct from the vacuoles, the latter
being indicated by the more lightly shaded portions of the protoplasm.
It will also be noted that from the first the micronucleus, or blepharoplast,
is closely attached to the eosin body (called by Leishman “ flagellar body ”).
Further, on the second day in this acid culture a few of the early
flagellated forms shown in figs. 7 and 8 of Line II were also seen, although
they do not usually appear in alkaline cultures until at least the third day,
while just antecedent to this stage are the forms shown in figs. 5 and 6
of the same line, illustrating commencing elongation and division by fission,
and it will be observed that in these the eosin body is passing up to the
anterior end of the organism from which the flagellum arises, and is carrying
the micronucleus with it. In my earlier description I suggested that the
double elongated forms shown in fig. 6 of this line might possibly represent
a form of conjugation preparatory to the development of the flagellated
stage of the organism, but further study of a much larger amount of material
has convinced me that they are only fission forms, as I have been unable to
make out any reduction in the number of chromosomes in the macronucleus
during the process.
The Mode of Division of the Flagellated Forms and the Formation of Rosettes.
In Line III of the plate are represented the different stages of division of
newly formed flagellated bodies. Figs. 1 and 2 show that the micronucleus
and flagellum first divide, just as in trypanosomes, and next the macro-
nucleus divides in turn, and a clear line appears in the length of the
organism, indicating commencing division of the protoplasm of the body, as
1905.| Kala-Azar, etc., from Leishman-Donovan Bodies. 287
shown in figs. 5 to 5 of the same line; while in fig. 6 the division of the body
has just been completed, and in fig. 7 the micronuclei and flagella of a still
adherent pair are dividing over again, thus showing how rapidly the
multiplication was taking place in this culture, for such forms were not
uncommonly seen in it.
In my earlier cultures the flagellated pairs were nearly always found in
pairs only, although rarely three or four might be seen side by side. In the
much more abundant development of flagellates in the acid culture medium,
however, considerably larger masses, forming beautiful rosettes, with the
flagella crossing each other in the centre, were seen in large numbers, and it
is easy to understand how they may be formed by the rapid multiplication
just described. Thus fig. 6 of Line IV shows a small group of flagellates
which is remarkable for including nearly all the stages of division in a single
clump, while fig. 9 shows the commencement of the formation of a rosette by
the rapidly dividing flagellates pushing each other round to form a semi-
circular mass, and in fig. 12 is shown a small, but complete, rosette, several
of the forms in which are undergoing further subdivision. In this stage the
contents of the eosin bodies frequently becomes protruded, as I have
previously noted, and it accumulates round the flagella, helping to bind the
forms together into the rosette shape. Next, the individual organisms
elongate, and at the same time become narrower, and the rosette then
commences to break up, in consequence of the increasing motility of the
flagella, and some now separate from the mass in pairs or single forms as
indicated in fig. 11, and in this manner the free swimming forms shown in
Lines IV and V of the plate are produced. In fresh specimens these are
very active, the single ones in particular threading their way rapidly among
the red corpuscles, and on reaching an open space, dart about in such a
manner as to leave no doubt in the mind of the observer that the object of
this remarkable development and extraordinary increase in size is to endow
the motionless human stage of the organism with the power of loco-
motion required in some period of its extra-corporeal existence.
The Nature of the Fully Developed Flagellate Form of the Organism.
When I first obtained the development of the flagellated stage, I thought
them to be young trypanosomes which had not yet formed an undulating
membrane. In support of this possibility, the recent observations of Novy
and MacNeil (12) on the culture of trypanosomes of birds on blood agar are
of great interest, for they obtained forms, separate and in rosettes, most
closely resembling those shown in the plate accompanying the present paper,
288 Dr. L. Rogers. Development of Hepatomonas of [Oct. 16,
both in the absence of all trace of undulating membrane and in the position
of the micronucleus or blepharoplast at the anterior flagellated end of the
organism, although in addition they obtained forms showing the development
of the membrane by the passage of the blepharoplast back towards, and
then past, the macronucleus, until it arrived near the posterior end of the
organism, and a typical trypanosome resulted. When further experience of
my culture failed to reveal any forms with a complete or even partial
undulating membrane, the question arose whether it was not an organism
distinct from the trypanosomes, although closely related to it, such as a
hepatomonas in which no undulating membrane is present. In my last
paper I left this an open question, while stating that nothing had yet been
found which might not be an incompletely developed trypanosome; a view
which has also been adopted by both Christophers (10) and Leishman (11),
The more abundant and uniform development of flagellates in the acidified
medium have enabled me to study closely innumerable apparently com-
pletely developed long free forms, in a stage in which they show extremely
active movement in fresh specimens: but still no trace of an undulating
membrane, or even a tendency for the micronucleus to pass away from the
anterior end of the organism towards the macronucleus has ever been
observed, although seen by Novy and MacNeil in their cultures of bird
trypanosomes. I therefore conclude that the organism I have been able to
develop belongs to the order Hepatomonas and not to the trypanosomes, and
I propose to name it the Hepatomonas of Kala-azar. At the ‘same time ii.
prefer to limit the term kala-azar to the epidemic-spreading form of the
disease as seen in Assam, and to retain the term “cachexial fever ” for the
less fatal sporadic affection, if only for the sake of avoiding the unnecessary
eruelty of having to tell sufferers from the milder disease that they are
suffering from the greatly dreaded kala-azar.
Degenerate Fors.
I have already pointed out that the absence of bacteria is necessary for
the continued development of the flagellated stage of the organism, and that
cocel especially are inimical to its growth. In one of my most active recent
cultures staphylococci gained access to the tube on the seventh day of the
culture during its repeated examination, and the degenerating changes
resulting were readily followed. On the following day fresh specimens
showed that all motion of the flagella had ceased, although on staining many
of the organisms showed little or no change. Others, however, were granular
and stained more lightly, while some were becoming shorter and more oval or
1905.| Kala-Azar, etc., from Leishman-Donovan Bodies. 289
pear-shaped, and their flagella shorter, as in figs. 2 to 6 of Line VI, thus
showing a tendency to reversion towards the undeveloped spleen stage of the
parasite, only all stages of the degenerative process were present at the same
time, and many of the shrunken badly staining forms were disintegrating.
During this process the flagella were often shed, and with it the micro-
nucleus came away, although a narrow non-staining space was still visible
between the two, as shown in fig. 10, clearly proving an organic connection
between the flagellum and the micronucleus or blepharoplast. Within three
days all the rosettes of flagellates had broken up into granular masses and
their identity completely lost. The degenerative changes in this hepatomonas
are therefore very similar to those which have been described in the case of
trypanosomes.
The Lelationship of Leucocytes to the Parasites in Cultures.
Although it is doubtful as yet whether the Leishman-Donovan bodies can
be found in the peripheral blood either free or in the red corpuscles, yet both
Donovan (+) and Christophers(13) have found this stage of the parasite
within leucocytes in the circulating blood during high fever, the latter
having twice found a number of them, nearly all within polymorphonuclears,
during a differential count of 500 leucocytes, which would mean an enormous
number within the peripheral circulation at one time, and amply sufhcient to
infect a blood-sucking insect if such proved a suitable host. It is therefore
of interest to determine if the parasites can develop in acid cultures within
leucocytes. Figs. 7 and 8 illustrate conditions bearing on this point, the
former representing a polymorphonuclear on the second day of the culture,
which contains typical parasites, although they are somewhat less developed
than those shown beneath it from the same slide: the latter shows another
degenerating leucocyte from the same culture on the following day, in which
some of the parasites are clearly much enlarged and developing typically, if
somewhat more slowly, than those show outside the corpuscle, while others
are degenerating and staining feebly. It appears then that development
may proceed within leucocytes, while Christophers is also of opinion that it
oceurs within macrophages in cultures, so biting insects might be infected by
the leucocytes containing the undeveloped parasites which have been found
in the peripheral blood.
The way in which the polymorphonuclears especially take up the parasites
in the peripheral blood is also of great interest in connection with the
extreme decrease in these corpuscles, for I have shown that they are
commonly decreased a tenth of the normal number, while in the latter stages
of the disease, in children especially, they may fall to only from one-
290 Dr. L. Rogers. Development of Hepatomonas of [Oct. 16,
twentieth to one-sixtieth, thus readily accounting for the frequency of
terminal infection by such diseases as dysentery, cancrum oris, pneumonia
and phthisis, owing to loss of phagocytic power, while I have also found
the opsonic index reduced against the staphylococcus pyogenes aureus, which
is frequently present in the spleen in cases of cancrum oris.
The Bearing of the Flagellation of the Parasite in Sterile Acid Medium on
the Probable Mode of Infection.
The two factors which I have found most essential to a uniform develop-
ment and very rapid multiplication of the flagellated forms are sterility and
a slightly acid, or, at least, a neutral medium. I have also tried blood agar
after Novy’s method, only using human blood im its preparation, but failed
to obtain either sub-cultures of already developed flagellates or of the spleen
parasites, while only very scanty development was obtained when several
drops of spleen blood, with very numerous parasites, were added to a
previously acidified blood agar tube, and then only in the added blood as
by the ordinary method. Now the only condition under which the
Leishman-Donovan bodies would be likely to meet with a sterile acid medium
on their escape from the human body would be in the stomach of some
blood-sucking insect, of which the common bed bug, or possibly mosquitos,
are the most likely hosts, for clinical reasons I have elsewhere pointed out,
while I have found that after sucking blood it becomes acidified in gastro-
ntestinal tract of bugs, and is also frequently sterile. I have not yet
succeeded in inducing these insects to suck infected spleen blood placed in
capsules of various kinds, but, on the other hand, I have mixed the contents
of their stomachs after feeding on human blood (which was proved to be
free from anything resembling any stage of the Hepatomonas of kala-azar)
with about an equal quantity of spleen blood containing the parasites, and,
after incubating in capillary tubes at 22° C., have been able to watch the
development of the parasites day by day up to the flagellated stage under
these conditions in those which remained sterile, but not when any bacteria
were present. It is therefore clear that the conditions met with in the
stomachs of bugs—and possibly also of mosquitos—are not inimical to the
development of the parasite into the flagellated stage, provided the
temperature conditions are suitable.
The more difficult question whether opportunities for infection of such
insects occur sufficiently frequently to account for the incidence of the disease
remains to be considered. In the first place, it is conceivable that bugs
especially might become infected from skin lesions containing the parasites,
for these may occur on parts of the body little exposed to the bites of
1905.| Kala-Azar, etc., from Leishman-Donovan Bodies. 291
mosquitos, but, in my experience such skin affections are too rare to alone
account for the frequency of infection. Further consideration will, I think,
show that the difficulty in finding these minute parasites in the peripheral
blood does not necessarily exclude the possibility of their occurring there
in sufficient numbers to infect insects, especially during high fever, when
they have been found in circulating leucocytes. In the first place, it has
been shown, by Christophers especially, that the organisms multiply in the
endothelial cells lining the blood-vessels of the internal organs, such as the
spleen, liver, and bone-marrow, and when numerous, in films obtained by
spleen puncture, they are frequently seen in groups in fragments of these
cells, which during life must frequently rupture and set them free in the
circulation, as is also proved by the same observer having found them in the
blood of some of the large veins. It is further of interest to note that the
endothelial cells of these very same organs are the principal sites of the
deposits of malarial parasites in the internal organs, while I have also
several times found Leishman bodies in the brain (where malarial parasites
also occur), so that it is clear that they must frequently enter the circulating
blood in considerable numbers. Secondly, the human stage of the parasite
is so small that it would be ‘scarcely easier to find in the blood by micro-
scopical examination alone that typhoid bacilli in that disease, although the
latter can be readily obtained by cultural methods.
The great difficulty of finding the human stage of the Hepatomonas of
kala-azar in the blood, even if present in sufficient numbers to infect
suitable insects, is well shown by Novy and MacNeil’s(12) experience of
searching for trypanosomes in birds; for while they only succeeded in
detecting this large actively moving parasite by microscopical examination
of thick blood films in 8 per cent., nevertheless they cultivated the parasite
on their blood-agar medium in 50 per cent. of the same series. Moreover,
even when they found them by their movement in thick fresh films, yet in
the same birds they frequently failed to detect them in stained specimens.
How much more difficult would it be to demonstrate the minute motionless
Leishman bodies, which can be only seen in thin stained films, even if they
were present in relatively large numbers in the peripheral blood ?
Thirdly, the extremely rapid multiplication of the flagellated forms in
some of my recent cultures would appear to indicate that, in the presumably
still more favourable natural conditions of the extra-corporeal stage of the
parasite, a very small number of the human organisms would multiply to
such an extent as to constitute a powerful infective agency.
The only reasonable alternative to the hypothesis just set forth is the
suggestion of Manson, Christophers, and others, that the organism may escape
292 Dr. L. Rogers. Development of Hepatomonas of {Oct. 16,
8 yi p
from the body by means of ulcers sometimes found in the intestines, the
granulation tissue ~f which contains the parasites, and they may thus
reach water. Apart trom the great rarity of such infected intestinal lesions
in my very extensive post-mortem experience of this disease in Assam and
Calcutta, the fact that sterility is essential for the continued development of
the flagellated stage of the organism appears.to me to make this mode of
infection an exceedingly improbable one. Moreover, I have been unable
to obtain any development of the organism in even sterile water kept at the
most favourable temperature, while even in sterile acidified water similar
negative results have recently been obtained.
Relationship of the Optimum Temperature for the Development of the Flagellates
to the Seasonal Incidence of Kala-Azar and Cachexial Fever.
If the conditions I have found necessary for the development of the
flagellate stage of the Hepatomonas of kala-azar afford any indication of the
natural conditions under which it occurs, then the striking fact that the
relatively low temperature of about 22° C., or 72° F., is essential to the
process, would indicate that infection is only likely to take place in India
during the colder part of the year. Owing to the fever in this disease
lasting for many months, or even several years, with long intervals of little
or no rise of temperature, while cases not infrequently begin very insidiously,
patients presenting themselves with marked, but often unsuspected, enlarge-
ment of the spleen, and a history of only a few days’ fever; it appears
probable that the incubation period may be a long one, and the onset very
insidious and indefinite. Nevertheless a clear history can often be obtained,
and an analysis of the notes of a number of cases showed five times as
many in which the symptoms first commenced in the six months from
November to April as in the remaining six hot months of the year, so that
the cold weather months, together with the very commencement of the’ hot
weather, to allow for the probable incubation period, show a very marked
preponderance of the infection. Moreover, Dr. Dodds Price, of Assam,
informs me, as a result of his unique experience of kala-azar, extending over
15 years, that every case he has seen in Europeans began in the cold season,
and that among his hundreds of native eases .he has noticed the same marked
tendency for definite symptoms of the disease to first show themselves at
that time of the year. The practical importance of this point in relation
to the prevention of the disease is evident, while the close agreement of its
seasonal incidence with the deductions from my experimental data is of
considerable interest. It is also worthy of note that this disease is most
prevalent in just those parts of India where the temperature conditions for
Roy. Soc. Proc., B. vol. 77, Plate 7.
1905.| Kala-Azar, ete., from Leishman-Donovan Bodies. 293
several months of the cold season most closely correspond with that which
I have found to be most favourable to the development of the flagellated
stage of the Hepatomonas of kala-azar, namely Assam, Bengal, and Madras.
On the other hand, the disease is much rarer, or has not yet been proved to
originate, in those parts of India where the winter season presents a
greater degree of cold, and the more favourable spring and autumn are
very short.
Much work will be necessary to test the truth or otherwise of the above
hypothesis, but knowledge should mean power to prevent the most terrible
of all tropical diseases in its combined very high mortality and slow death
by inches, and as the most favourable cold weather working season is
approaching, it appears to be advisable to put these observations on record
for the benefit of other workers in this very important field of tropical
medicine.
REFERENCES.
1. Leishman, “On the Possibility of the Occurrence of Trypanosomiasis in India,”
‘Brit. Med. Journ., vol. 1, 1903, p. 1252.
. ‘ Brit. Med. Journ.,’ vol. 2, 1903, p. 79.
. ‘ Bull. de Acad. de Med.,’ vol. 1, p. 288.
. “haneet,’ vol. 2, 1903, p. 44.
. ‘Brit. Med. Journ., vol. 2, 1903, p. 1401.
. ‘Scientific Memoirs, Government of India,’ No. 18, New Series.
. * Brit. Med. Journ.,’ September, 1904.
. ‘Quart. Journ. of Micro. Sci.,’ November, 1904.
. ‘Lancet,’ vol. 1, 1905, p. 16.
10. ‘Scientific Memoirs, Government of India,’ No. 15, New Series.
11. ‘Journ. of Roy. Army Med. Corps,’ March, 1905.
12, * Journal of Infectious Diseases,’ vol. 2, 1905, p. 256.
13. ‘Scientific Memoirs, Government of India, No. 11, New Series.
to
w
IOS oO
co oO
DESCRIPTION OF PLATE,
Magnification of all the figures 1500 diameters.
Iie. 1.—Undeveloped Leishman-Donovyan bodies from spleen puncture film.
“4 Il.—Early stages of development, from two days’ culture in acidified citrated blood ;
1 and 2, body and macronucleus enlarged ; 3 and 4, first appearance of eosin
body; 5 and 6, elongation and subdivision ; 7 and 8, first appearance of
flagellin.
» II1,Stages of division of the early flagellated forms.
» LV.—Double long swimming forms.
“r V.—-Fully developed long, free, active single cells.
» Wi.—Degenerate forms.
. WIl—Undeveloped forms in a white corpuscle.
» VIII-—Early stages of development in a degenerating white corpuscle.
, IX.—Stage in the formation of rosette.
» X.—Separated flagella with micronuclei attached.
» XI.—Rosette breaking up into free forms.
, XNL£I.—Small complete rosette.
294
The Factors which Determine the Production of Intraocular
Fluid.
By E. E. HENDERSON and E. H. Srarwine, F.R.S.
(From the Physiological Laboratory, University College.)
(Received November 23, 1905.—Read January 18, 1906.)
In spite of the very numerous researches which have been made during the
last half century on the seat and mechanism of production of intraocular
fluid, ophthalmologists and physiologists are still far from an agreement on
the subject, and a review of the literature reveals many discrepancies in the
experimental evidence which it is impossible to clear away without a
re-examination of the whole subject. The following paper contains the
results of experiments made with the view of determining the weight to be
ascribed to different experimental investigations.
As to the seat of production of the intraocular fluid, nearly all authorities
are agreed that it is produced by. the ciliary processes. From these processes
a minute proportion travels backwards into the vitreous cavity, to be absorbed
by the lymphatics of the optic disc, while by far the greater part makes its
way between the lens and the ciliary processes, through the fibres of the
suspensory ligament, into the posterior chamber, whence it passes round the
margin of the iris into the anterior chamber. In addition to this mode of
production, it has been suggested by Ehrlich that an appreciable amount of
intraocular fluid may be secreted directly into the anterior chamber by the
anterior surface of the iris. The experiments of Ehrlich(1) were made by
the injection of a diffusible substance, fluorescine, aud we agree with Leber (2)
in regarding them as proving the possibility of diffusion between the vessels
in the iris and the anterior chamber, but not the secretion of a normal intra-
ocular fluid by this channel. At any rate, any fluid formed in this way is
negligible when compared with that which is produced in the neighbourhood
of the ciliary processes.
On the other hand, the place of absorption of the intraocular fluid is
universally agreed to be the angle of the anterior chamber. Here the fluid is
passed under pressure into the spaces of Fontana, whence it makes its way
into the canal of Schlemm, between the endothelial cells lining this canal, and
so is carried away into the venous system. This absorption is continuous,
and its rapidity is largely determined by the height of the intraocular
pressure. Since we have a constant absorption and a constant pouring out
of fluid into the eyeball, it is evident that the intraocular pressure must be
Factors which Determine Production of Intraocular Fluid. 295
a product of the two factors, formation and absorption, and that the main-
tenance of the pressure at a constant height must be determined by an
accurate balance between these two processes. The problem which lies
before us is to determine the mechanism of formation of this fluid.
The intraocular fluid is a clear, colourless solution containing a proportion
of salts similar to that of the blood plasma, but having an osmotic pressure
which is somewhat higher than the blood plasma, and containing the merest
trace of proteids.*
I. Methods of Research.
The animals used were mostly cats. In a few cases dogs were employed,
and in one experiment a rabbit. In the case of the cats the anesthetic
used was always ether, with the addition in some cases of a small dose of
morphia. In a few experiments, after the induction of full anesthesia,
a small dose of curare was given. The administration of the anesthetic was
continued during the experiment by an air-pump connected with a cannula
in the trachea. For the dogs the A.C.E. mixture was employed.
A record of the blood pressure was kept in all experiments. In some it
was taken continuously, but in the greater number of experiments a short
record was taken every few minutes in order to avoid trouble with clotting
in the cannula. In the cats the blood pressure was taken in the lower part
of the abdominal aorta, in the dog in the femoral artery.
The apparatus we employed for measuring the intraocular pressure was
very similar to that described in a former paper (3). A graduated tube with
internal bore of about 0°5 mm.,and about 50 em. long, is provided with
a lateral tube near each end. One end of the tube is connected by india-
rubber tubing, by means of a T-piece, with a reservoir containing Ringer’s
solution (or any other fluid the absorption of which is to be determined), and
also with a manometer. The other end is connected by a second (glass) tube
with a gilt steel hollow needle, which is introduced into the anterior chamber
of the eye. The needle may be open at the end, or be closed at the end and
provided with a lateral opening. To each of the side tubes a rubber capsule
is attached. The capsule nearest the reservoir contains air, while that
towards the eye is filled with fluid. By means of screw-clamps, fluid or air
may be driven from either of the two capsules into the graduated tube.
Before introducing the needle into the anterior chamber, the pressure in the
apparatus is adjusted by raising the reservoir to about 25 cm. H20, which
represents the average intraocular pressure. While the fluid is dropping from
* Full details of various analyses of intraocular fluid are given by Leber (2), p. 207,
et seq.
296 Mr. E. E. Henderson and Prof. E. H. Starling. [Nov. 23,
the end of the needle, this latter is thrust through the lateral part of the
cornea, so as to lie in the middle of the anterior chamber. A bubble of air
is introduced into the graduated tube by compression of one capsule. and
brought to the middle of the tube by relaxing the clamp on the capsule at
the end towards the eye. The reservoir is then rapidly adjusted to such
a height that the bubble remains stationary.
In some of the later experiments a platino-iridium cannula, with a solid
steel point made slightly conical, was found to be an improvement, as, in the
event of any leaking occurring, 1t could be pushed in further.
In introducing the cannula great care must be used, as, should the needle
catch in or tear the iris, or wound the lens, the eye would be rendered useless
for the purposes of the experiment. The needle, being comparatively large
and blunt, requires considerable force for its introduction. We have found
it safer to make a small perforation with the point of a cataract knife and,
without letting the aqueous humour escape, to introduce the cannula in the
hole thus made. Should the exact spot be lost sight of, a little fluorescine
will stain it. A fine silk thread passed through the episcleral tissue, as in the
operation for advancement of a rectus tendon, gives a better hold than fixation
forceps, and is somewhat less in the way.
The fluid employed in the apparatus was usually Ringer’s solution, in some
cases normal saline. Whichever fluid was employed, it was filtered through a
Berkefeld candle before the experiment, in order that no foreign body might
be present which could lodge in and block the filtration channels.
The intraocular fluid must play a twofold function in the eye. In the
first place, by keeping up the intraocular pressure, it lends rigidity to the
supporting structures of the eyeball, and furnishes therefore a fixed point for
the intraocular muscles to contract against, besides maintaining the proper
distances between the various refractive media. In the second place, it is
the only source of nourishment to certain of the structures of the eye,
namely, the middle and back part of the cornea, the lens and suspensory
ligament, and the vitreous humour. The question that we have to decide
is whether this fluid is formed by a process of secretion by the cells covering
the ciliary processes, or whether it is a transudation similar to lymph. The
question presents many analogies to that with regard to the secretion of urine.
In each case we have a possible source of transudation in the capillary
blood-vessel network and also an absorbing mechanism. We can only arrive
at a conclusion by determining the physiological conditions under which
we may alter either the production or the absorption of the intraocular
fluid.
1905. | On the Production of Intraocular Fluid. 297
Il. The Effect of Changes in the Circulation on the Formation of Intraocular
Fluid.
If the production of intraocular fluid is dependent on a process of filtra-
tion through the blood vessels and the epithelium covering the ciliary
processes, its rate must vary directly with the difference of pressure on the
two sides of the filtering membrane. It must vary, therefore, directly with
changes in the capillary blood pressure, and inversely with the changes in
the intraocular pressure. In our first series of experiments we sought to
eliminate the second factor, namely, that of absorption, by opening the
anterior chamber, so that the intraocular pressure could be regarded as zero.
A cannula was introduced into the anterior chamber and the fluid allowed
to flow off into weighed porcelain capsules. These were changed every
10 or 20 minutes, and the amount of fluid secreted in the time determined
by weighing. The fluid drained off during the first minute after insertion of
the cannula was regarded as normal intraocular fluid, but the gradual
emptying of the eye-ball continues during the first five minutes, so that the
figures obtained during this time cannot be regarded as expressing the rate
of secretion. In every case the total solids of the intraocular fluid were also
determined.
The following experiment, p. 298, shows the results obtained while the blood
pressure was approximately constant. It will be seen that there is a constant
diminution in the amount of fluid obtained. In these experiments we were
at first troubled by the formation in the anterior chamber of clots, which
tended to plug the cannula. We found that this difficulty could be obviated
by the injection of a dose of leech extract, not large enough to cause a
permanent diminution of the blood pressure.*
The next question to determine was whether it was possible to alter the
rate of production or the composition of the intraocular fluid by altering the
blood pressure in the vessels of the eye-ball. The experiments on this point
were all carried out on dogs. A diminution of the intraocular blood pressure
was easily effected by ligature or obstruction of the carotid artery on the same
side. In order to produce a maximal rise of pressure in the blood-vessels of
one eye, the vertebral and subclavian arteries on both sides were tied.
A loose ligature was placed round the thoracic aorta, so as to permit of its
being obstructed at any given time. A cannula connected to the mercurial
manometer was placed in the carotid artery on the right side. The production
of intraocular fluid was determined in the left eye. By obstruction of the
* This procedure had been previously employed by Mr. E. Pfliiger in some experiments
carried out in this laboratory in 1902. An account of these experiments will shortly
appear.as a dissertation in the University of Bern.
298 Mr. E. E. Henderson and Prof. E. H. Starling. [Nov. 238,
aorta a large rise of blood pressure was produced in this eye, since all the
blood had to pass through the one carotid artery in order to get back to the
heart. On the other hand, an almost complete anemia could be produced in
the eye by obstruction of the one remaining carotid. We give below the
results of one such experiment.
Cat, anesthetised with Ether and the A.C.E. Mixture. A small dose of
Curare was injected after anesthesia was complete. The extract of
2 grammes of dried leech heads was injected.
| Weight of
; c lids aft
Time. B.P. » mm.| Weight of are Percentage | Rate of flow
g. secretion. Borisbanit of solids. | per minute.
| weight.
grammes. | grammes. grammes.
3.50 cannula inserted.
Bai ososntesaossoosuanaeos ~ 130 0-689 0-009 1°3
BHAT essd00 594 egae09000800500 145 0:252 | 0-007 2-7 0:05
GBI) 4a: nanSBaceueeoose 120 0-756 | 0°032 4,°2 0-037
A BG Ns jrctuee weasanen denise 100 0-475 0-021 44 0-023
ANS Greatecetccmesecceceoscses 96 0 482 0-024 4°9 0-024
Dog. Weight, 73 kilos. Anzsthetised with the A.C.E. mixture and morphia.
The extract of 2 grammes of dried leech heads was injected. Both
subclavians and vertebrals were tied. Temporary ligature round aorta.
Cannula in left eye. 3B.P. observed in right carotid.
- Amount of Total
Time. oe : secretion in | solids in eecntsee pra Remarks.
grammes. | grammes.
3°29 —_ — — — — Cannula inserted. Aorta
unobstructed
3°30 110 0°811 0-013 1°5 —
3°35 110 0 *432 0°014 3:2 “086
3°45 100 0 ‘550 0 027 4:9 0-055
BEDD 205 1°153 0-068 5°9 0-115 | Aorta obstructed. Fluid
tinged red.
4°5 100 0 ‘627 0 ‘039 6:2 0-062 | Aorta unobstructed.
4°15 198 0°816 0-053 6°6 0-081 | Aorta obstructed.
It will be seen that in every case a rise of intraocular pressure caused an
increase in the amount of fluid secreted. It is impossible, however, to deduce
directly from these experiments that the intraocular fluid is a transudation.
The opening of the eye-ball and the consequent diminution of the intraocular
pressure to nothing have a serious effect on all the intraocular structures.
1905. | On the Production of Intraocular Fluid. 299
Great dilatation of the vessels of the ciliary processes and iris is produced.
The fluid, which, in the normal eye, is free from fibrinogen and contains the
merest trace of proteid, rapidly acquires the power of coagulation, and its
proteid content rises to 3, 4, or 5 per cent. The serious alteration of the
vascular structures is shown in many cases by the appearance of red blood
corpuscles in the fluid dropping from the cannula, and Greeff has shown that
if the lowered pressure be brought about suddenly and maintained for some
time, the epithelium covering the ciliary processes may be raised from the
surrounding tissue so as to form small blisters, which are filled with coagul-
able lymph. It has been suggested by Greeff (4) that the change in composi-
tion of the intraocular fluid ensuing on opening the eye-ball is determined
by the separation of the epithelium, but Bauer (5) has shown that the proteid
contents may be raised in the absence of these epithelial changes, and that,
on the other hand, the epithelial changes may be well marked on the
subsequent day, when the wound in the cornea has closed, and the intra-
ocular fluid has regained its normal composition. He also points out that
the amount of change produced depends entirely on the rapidity with which
the intraocular pressure is lowered. The change in composition is probably
due, as Leber suggests, to the great distension of the capillaries and the
consequent separation of their endothelial cells. It represents in fact an
alteration in permeability of the filtering membrane.
II. Amount of Intraocular Fluid Produced under Normal Circumstances.
Im any investigation of the factors determining the production and absorp-
fion of intraocular fluid, it is important to get some idea of the amount of
this fluid secreted under normal circumstances, that is at normal intraocular
pressure, Since the intraocular pressure is maintained constant so long as
the blood pressure is steady, the amount of fluid produced at a given intra-
ocular pressure must be equal to the amount of fluid absorbed at the same
pressure. It is therefore a matter of indifference whether we measure the
amount formed or the amount absorbed at any given pressure. Le Plat (6)
sought to abolish the absorption of the intraocular fluid by filling the
anterior chamber with oil or vaseline. A cannula was placed in the vitreous
cavity, and the pressure in the cannula maintained at the normal intraocular
pressure. It was found that the obstruction of the absorbing angle of the
eye-ball carried out in this way caused a rise of intraocular pressure if the
eye-ball were closed, or a flow outwards of intraocular fluid by the cannula
if the pressure in this was maintained at the normal intraocular pressure.
The amount of this outflow was measured, and was regarded by Le Plat as
representing the normal rate of formation of intraocular fluid. He arrived at
VOU) Evil: ¥
300 Mr. E. E. Henderson and Prof. E. H. Starling. [Nov. 23
the conclusion that the amount of fluid normally secreted by the ciliary
processes is in the rabbit about 4 c.mm. per minute. We found considerable
difficulties in applying this method, chiefly determined by the tendency
of the cannula in the vitreous to become blocked. We therefore adopted
a method similar to that already employed by Niesnamoff, (7) under Leber’s
direction. The arrangement of the experiment was as follows :—
The hollow needle, connected by the capillary tube (containing an air
bubble as index) to the reservoir and manometer, was introduced into the
anterior chamber. The height of the reservoir was then adjusted until the
bubble was stationary, showing that the intraocular pressure was exactly
balanced by the pressure of the fluid in the tube leading to the reservoir.
This intraocular pressure was of course maintained by a constant secretion
of intraocular fluid, exactly equal to the amount escaping by filtration through
the anterior angle of the eye. The animal was then killed by dividing the
heart. This procedure at once stopped the production of intraocular fluid.
The intraocular pressure, however, was maintained at its previous height by
the connection of the eye with the reservoir of Ringer's fluid; the escape
fluid by the anterior angle was therefore the same as before. The rate of
this escape could be determined by noting the rapidity with which the
air bubble moved along the capillary tube towards the eye, and this rate
must be equal to the rate of production of fluid previously obtaining in the
eye under normal conditions of circulation. The following table gives the
rate of production of intraocular fluid, determined in this way, with varying
intraocular pressures :—
Intraocular Inflow, after cessation of
Animal, pressure in mm. circulation, in cubic
Hg. millimetres per minute.
(Oy repyacdooucopacdeds 20 12
Cater Aicdamieaine: 15 1l
(CE bash. cosemboannadose 26 12
(Ohieoonaoesseqnabdand 28 10
(Giitpasseacosesanagsa 14 5
(Ohh regannpenpancacsn 20 15
Average...... 20°35 10°8
It will be seen that there is a considerable difference in the case of filtra-
tion in various eyes, and therefore a corresponding difference in rate of
production of intraocular fluid,
1905. | On the Production of Intraocular Fluid. 301
IV. The Factors Determining Absorption of Intraocular Fluid.
In the last set of experiments we determined the rate of absorption of
intraocular fluid at the normal intraocular pressure, and regarded this as
representing the rate of production of this fluid under normal circumstances.
In the same experiment it was possible to alter the intraocular pressure by
raising or lowering the reservoir, and so to determine the effect of the height
of the intraocular pressure on the rate of absorption. The results of two
such experiments are given below, and show conclusively that the rate of
absorption is determined, in the absence of disturbing factors which we
shall have to consider later on, solely by the height of intraocular pressure.
(1) Cat, anesthetised with Ether. While the anesthesia was maintained, a
small dose of morphia and curare was injected. Atropine was instilled
locally into the conjunctival sac.
Rate of inflow in cubic
B.P. in mm. Hg. | 1.0.P. in mm. Hg. millimetres per minute.
115 22 10)
115 30 4
115 46 a
130 62 8
0 22 12
0) | 36 16
0 46 19
0 5
(2) Cat, aneesthetised with Ether. Atropine and cocaine instilled locally into
the conjunctival sae.
B.P. in mm. He. | 1.0.P.in mm. He. Hes @ aalllos tn OL
millimetres per minute.
124 32 0)
124: 44. 5)
12 52 am
110 20 0)
116 Ad 10
116 52 20
Heart divided.
10) 52 22
0 44. 15
0 20 12
302 Mr. E. E. Henderson and Prof. E. H. Starling. [Nov. 23,
In a previous paper we have shown that the intraocular pressure varies
directly as the blood pressure in the vessels of the eyeball. We must
therefore conclude that the rate of absorption of intraocular fluid is also
determined by the height of the blood pressure, and since the absorption
must keep pace exactly with the formation of this fluid, it follows that the
formation of the intraocular fluid must also be determined by the height of
the intraocular blood pressure. So far then the conditions which we laid
down as necessary to be fulfilled in order to justify the filtration theory of
the production of intraocular fluid have been fulfilled, and we might conclude
with Leber that the formation of this fluid is exactly analogous to that of
lymph, and is determined by the difference of pressure between the blood in
the vessels and the fluid outside the vessels. There are, however, certain
difficulties in this assumption which have so far not been considered by
previous workers, but which must be met satisfactorily before we can
come to any definite conclusion on the subject.
Tt has hitherto been assumed by Leber, Niesnamoff, and others, that a fluid
having the composition of intraocular fluid might be formed by a process of
filtration through the blood vessels of the ciliary processes under any
difference of pressure. In this assumption they have neglected the question
of the different proteid content of blood plasma and intraocular fluid. It was
shown by one of us (EK. H. 8.) that, in order to separate a proteid-free
transudate from a fluid such as blood serum, a certain amount of work had
to be done, and that for this separation a minimum difference of pressure on
the two sides of the filtering membrane of at least 28 mm. Hg was
necessary. The intraocular fluid has such a small content in proteid that
it may be regarded as analogous in all respects to the fluid which is supposed
to be separated vy the glomeruli of the kidney. In order therefore that any
fluid shall be poured out in the eyeball, a minimum difference of 30 mm. Hg
must be present between intraocular pressure and capillary blood pressure.
If this pressure difference is not present, work must be done by the cells
forming the filtering membrane, and the formation of intraocular fluid must
be regarded in the light of a secretion rather than in that of a transudation.
A definite decision on this point could be reached if we had any means of
determining the blood pressure in the capillaries of the eyeball. A method for
this purpose has been devised by Niesnamoff, (7) and this observer states that
the normal intraocular capillary pressure is about 50 mm. of mercury. His
arguments, however, involve several fallacies. In his experiments he con-
nected a cannula, attached to a reservoir of salt solution, with the eyeball
of a living animal. He found that the fluid neither ran in nor out at
25 mm. Hg, which was therefore the intraocular pressure. He then
1905. | On the Production of Intraocular Fiud. 303
determined the rate of inflow when the pressure in his cannula was raised
to 50 mm., 75 mm., and 100 mm. He. He then killed the animal, and again
determined the rate at which the fluid would flow in under these various
pressures. He found that above 50 mm. Hg the rate of inflow was the same
in the dead as in the living animal. He therefore concluded that 50 mm. Hg
represented the intracapillary pressure. In coming to this conclusion he was
guided by the assumption that, when the intraocular pressure was raised so as
to be equal to the intracapillary pressure, the transudation of intraocular
fluid would cease, and above this pressure the rate of inflow for his reservoir
would be, therefore, the same in the living and dead eye. It is impossible,
however, by this method to determine intracapillary pressure. The globe
of the eyeball is practically mgid. As the intraocular pressure is raised,
the intraocular fluid will press upon the veins of the ciliary processes, and
the blood pressure will therefore rise in the capillaries and in the veins
until it is greater than the intraocular pressure. With successive rises in the
intraocular pressure the pressure in capillaries and veins must get larger and
larger in order that any circulation of blood may be maintained, and the
circulation through the capillaries will cease only when the intraocular
pressure is very nearly as high as the arterial pressure. If the circu-
lation in Niesnamoff’s experiments ceased at 50 mm. Hg, it is evident
that the normal intracapillary pressure, when the intraocular pressure is
25 mm. Hg, must be considerably below 50 mm. Hg. How then are we
to explain the very definite figures obtained by Niesnamoff? This observer
apparently performed very few experiments. In his paper he gives the
results of only one such experiment as that here described. On repeating
his experiments we found it impossible to obtain anything like such definite
figures—and this for various reasons. In the first place, a considerable rise
of intraocular pressure, such as to 50 or 70 mm. Hg, exercises an abnormal
stretching effect upon the filtering apparatus of the eyeball, so that the
channels at the anterior angle of the eye are gradually opened up, and in
many experiments we observed a consequent gradual increase in the rate
ot inflow of the fluid. In most experiments, for example, the rate of inflow
was greater with descending pressures than with ascending pressures. This
is well shown in experiment No. 2, on p. 301.
The following experiment shows the dilatation consequent on a preliminary
raising of the intraocular pressure :—
304 Mr. E. E. Henderson and Prof. E. H. Starling. [Nov. 238,
Cat, anzesthetised with Ether. serine applied locally to conjunctival sac.
Pupil moderately contracted.
BP. in mm, Hg, | 1L.0.P. in mm. ag. eee eee eta
|
110 | 16 )
110 | 32 5
110 | 48 8
108 | 64 9
112 16 0)
112 32 §
112 48 13
| 112 64 18
Another disturbing factor is the size of the pupil. We shall have to
consider this factor more in detail later on, but unless atropin be given at
the beginning of the experiment, the observations on the living eye are made
with a somewhat contracted pupil, whereas those on the dead eye are
made on a widely dilated pupil. Other factors being equal, the filtration in
the eye with dilated pupil is always slower than in the eye with contracted
pupil. In certain of our experiments we observed an equality of inflow
between the dead and living eye at some pressure above 40 mm. of
mercury, but on further raising the pressure this equality disappeared,
showing that we were dealing with yielding tissues and altering membranes.
This fact rendered it impossible to obtain by such methods any definite
information of the intracapillary pressure in the eye-ball, or of the
level of intraocular pressure at which transudation or formation of intra-
ocular fluid would definitely cease. One other factor which would aid
in disturbing the results obtained is the effect of a high intraocular
pressure on the general circulation through the eye-ball. If we succeed in
raising the pressure to such a height that the circulation is entirely abolished,
changes must rapidly take place in the apparatus both for formation and
absorption of intraocular fluid, and subsequent results cannot be compared
with those obtained before such a cessation of circulation. The raising of the
intraocular pressure in itself may act as a stimulus and cause reflexly
alterations in blood flow, in the general blood pressure, or in the state of
contraction of the pupil. The co-operation of these various factors suttices to
explain the varying results obtained in the very many experiments we
performed upon this subject, including those of which we have already given
details. We are of opinion, therefore, that the results obtained by
Niesnamoff must be regarded as accidental, and that a greater number of
experiments would have convinced this observer of the fallacies of his method.
1905. | On the Production of Intraocular Flind, 305
Although it is impossible at present to determine the intracapillary
pressure in the ciliary processes, we may at avy rate inquire whether there
is, in all experiments on the subject, the possibility of a difference of pressure
of 30 mm. He between intracapillary blood pressure and intraocular pressure.
In the case of a similar question in the kidney, we have been accustomed
to compare the aortic blood pressure with the ureter pressure, and have
regarded a difference of 40 mm. between these two pressures as satisfying
the necessary conditions for filtration through the glomeruli. A similar
comparison of arterial blood pressure and intraocular pressure leads to the
same result. Below we give the intraocular pressure and arterial pressure
as determined in a series of 20 experiments. It will be seen that in
every case there is a difference between the two pressures of at least
48 mm. Hg, the average difference of pressure in all the experiments being
84:8 mm. He.
=e :
Animal. BE ‘He yates a eaees Lae eps sete sb
g. Ife.
Caltinscaenehiecsnccrns 1380 16 114
Gatien cdastises 140 25 115
(ODirocaseacesusostonde 188 20 118
Cate atm Asce snes 94 24 70
IRON Sonoposodone 74 16 58
ID ays: topdagaceseeee 112 14 98
Gath eae a des cbs 104 15 89
Watiecescaciaascaees 106 19 87
Cat 106 18 88 |
Cath tieschicssaecasien 120 20 100
(OD Hbchase fae Sap seReod 150 22 128
IDL Coanonceouaance 84. 12 72
Dog 58 10 48
Doge ered 70 16 54
(Ob) ibecoecodcesbnaded 115 23 92
Caterers sionen 12 32 92
Gata deve eceaet ca 110 16 94,
Catissanccneessnce nse 138 22 116
Cate aaiiae comic 94 27 67
Catisanasaacgeoueaeeceh 110 24 96
So far then our observations tend to support in every particular the view
laid down by Leber, namely, that intraocular fluid is produced in the ciliary
processes by a process of filtration, and that the sole factor determining the
amount of transuded fluid is the difference of pressure between the blood in
the capillaries and the fluid in the eye-ball.
V. Influence of the Proteid Content of the Intraocular Kluid on the Intraocular
Pressure.
The fact that the intraocular fluid has to be filtered through the intercellular
channels of the endothelium bounding the spaces of Fontana and lining the
306 Mr. E. BE. Henderson and Prof. E. H. Starling. [Nov. 23,
canal of Schlemm, in order to escape from the eyeball, suggests that the
resistance will be greater if the viscosity of the filtering fluid be increased in
consequence of raised proteid content. Indeed, one form of raised intra-
ocular pressure, the glaucoma accompanying inflammation of the ciliary
region, has been ascribed to the greater proteid content of the intraocular
fluid secreted by the inflamed vessels, and the consequent greater resistance
to the filtration of this fluid through the anterior angle of the eye. So far as
we are aware, there are no direct determinations of the relative rates of
filtration of normal salt solutions with and without proteid. We have,
therefore, in a series of animals, determined the intraocular pressure under
the two conditions :—
(a) With normal intraocular fluid.
(b) After replacing this fluid by blood serum.
We have also compared the relative rates of filtration of normal salt.
solution and of serum in the living and dead eye.
In our experiments one eye of the animal was connected with a reservoir
and manometer containing Ringer’s saline fluid, while the other was connected
with a similar apparatus filled with filtered blood serum.
In order to determine the intraocular pressure in an eye, in which the
normal aqueous humour had been replaced by serum, after introduction of
the hollow needle, the aqueous was allowed to escape through the side opening
in the cannula. Serum was then allowed to flow in for a time, and then the
contents of the anterior chamber again allowed to escape. The side tube was
then closed, an air bubble introduced into the capillary tube, and the pressure
determined at which the bubble moved neither backwards nor forwards.
In nearly every experiment the intraocular pressure, during the first 5 or
10 minutes after the insertion of the cannuia, was higher in the eye filled
with serum than in the eye filled with normal fluid. The difference, however,
rapidly diminished, so that 15 to 20 minutes after the beginning of the
observation the pressures were practically identical in the two eyes, and
remained so throughout the rest of the experiment. It must be remembered
that with the zero method used by us there is no movement of fluid into the
eye. Hence the fluid necessary to replace the loss by filtration and to
maintain the intraocular pressure is being constantly secreted by the ciliary
processes, and is probably of the normal composition, 7.c., practically free
from proteid. We should therefore expect a gradual decline of the intra-
ocular pressure in the eye with serum, although hardly so rapid an
equalisation of the pressures on the two sides as we actually observed in
our experiments,
1905. | On the Production of Intraocular Fluid. 307
After the determination of the intraocular pressure, the animal was killed
by opening its heart, and the inflow of serum and saline fluid respectively
observed, first under the normal intraocular pressure, and then under raised
pressures.
The results of two such experiments are given below. It will be seen that
there is a marked difference in the rate of filtration of the two fluids, that of
serum being, as one might predict, very much slower than that of saline.*
Experiment 1—Dog, A.C.E. Morphia. Curare. _ Vagi cut.
| Intraocular pressure.
Time. | Blood pressure. ——— =
| Salt eye. Serum eye.
4.15 70 mm. Hg. | 26 °2 29 -4 em. water.
4.20 70 i | 24-2 27 5
445 100 5 29:2 29 5
Animal lulled | by opening heart.
Inflow per minute in cubie milli-
metres (after 10 minutes).
Pressure. |
| ]
| Salt. Serum.
|
29 cm. | 11°5 6
oa 1a 59 } 6
| — | 1:5 | 6
Experiment 2.—Cat. Ether, morphia, curare.
| | Intraocular pressure.
Time. Blood pressure. or ta j
| | Salt. Serum.
| '
ar .
3.0P.M. | 120 148 15-1
S10 116 10°8 125
ly si20 | 110 9°2 me
| Animal Killed. |
* Although serum filters more slowly than normal intraocular fluid or saline, the
difference is not sufficiently great to cause any marked variation in the intraocular
pressures on the two sides. One cannot, therefore, in view of these observations, ascribe
any large part in the production of any form of glaucoma to possible differences in
the composition of the aqueous humour which might be determined by inflammatory
conditions of the blood vessels.
VOL. LXXVII.—B.
N
!
308 Mr. E. E..Henderson and Prof. E. H. Starling. [Nov. 23,
Inflow three minutes later at same intraocular pressures—
Salt. Serum.
6 3
5 3°5
5 6
i) 4
A 4
Fifteen minutes later—
3:5 15
30 15
Bi) 1G
ete ete
This difference in the rate of filtration of the two fluids becomes greater
the higher the intraocular pressure is raised.
VI. The Effect of the Size of the Pupil on the Absorption of Intraocular
Fluid.
In the experiments we made to decide this point, one eye of the animal
under observation was treated with eserine and the other with atropine.
The instillation of these drugs should be begun before the induction of
anesthesia, as the action of eserine is very uncertain if only instilled after
anzesthesia.
We have found, as a result of these experiments, that the intraocular
pressure in the two eyes remains the same during the time of observation,
but that, if the pressure in the apparatus be raised, the rate of filtration in
the eye under eserine is much greater than in that under atropine.
It is difficult to give a precise explanation as to the cause of this difference.
Stretching of the filtration spaces at the angle of the anterior chamber may
possibly account for it all. If this, however, is the case, we should expect to
find the intraocular pressure at a lower level in the eye with the contracted
pupil, for the intraocular: pressure must of course be the product of the rate
of secretion and the rate of absorption of the intraocular fluid. The same
objection applies to the explanation of this phenomenon by Grénholm (9), who
states that in his opinion it is due to diminished intraocular secretion as a
result of the contraction of the intraocular vessels. It may also be possible
that at these raised pressures other channels of filtration are opened up—
such for instance as the surface of the iris. An important, perhaps the most
important, factor, however, must be the crushing of the dilated flaccid iris
1905. | On the Production of Intraocular Fliad. 309
into the filtration angle of the eye, thus causing a mechanical obstruction,
which will be more marked the greater the intraocular pressure. Hence the
smaller amount of filtration in the atropinised or dead eye with dilated pupil,
as compared with that in the eye which has been put under the influence of
eserine.
The figures of a typical experiment are given.
Cat, anesthetised with Ether. Blood pressure average 138 mm. Hg, with
only trifling variations throughout the experiment.
Rate of filtration in Rate of filtration in | Rate of filtration in
Intraocular pressure | eserine eye in cubic | atropine eye in cubic | atropine eye post-
in mm. Hg. millimetres per millimetres per | mortem, in cubic
minute. minute. | millimetres per minute.
20 6) 0 | 15
35 11 8 20
50 | 16 1 | 25
65 23 14. 31
|
Summary of Conclusions.
1. The intraocular pressure represents the pressure at which the rate of
formation of intraocular fluid is exactly balanced by its rate of escape
through the filtration angle of the eye.
2. The production of intraocular fluid is strictly proportional to the
difference of pressure between the blood in the capillaries of the eyeball and
the intraocular fluid.
3. No satisfactory method of measuring the intracapillary pressure in the
eyeball has been yet devised. The fallacies of Niesnamoff’s method are
pointed out. Judging, however, from a comparison of the arterial pressures
and the intraocular pressures in a large number of animals under different
conditions, there is probably always a difference between the intracapillary
pressure and intraocular pressure, which is sufficient to account for the
production of the intraocular fluid, without assuming any active intervention
on the part of the cells of the capillary walls or of the ciliary processes.
4. An increased proteid content of intraocular fluid slows its rate of
absorption in consequence of the mechanical hindrance of the proteid
to filtration.
5, Filtration, 2.e., the absorption of intraocular fluids, at high intraocular
pressures is favoured by constriction of the pupil and hindered by dilatation
of the pupil. The difference, however, is barely perceptible with normal or
low intraocular pressures.
310 Production of Intraocular Fluid.
The expenses of this research were defrayed by a grant from the Scientific
Grants Committee of the British Medical Association.
BIBLIOGRAPHY.
1. Ehrlich, ‘ Deutsche med. Wochenschr., No. 2, ff., 1882.
2. Leber, ‘ Graefe-Saemisch, Handbuch der Gesam. Augenheilkunde, vol. 2, pt. 2, 1903.
3. Henderson and Starling, ‘Journ. of Physiol.,’ vol. 31, pt. 3, 1904.
4. Greef, ‘ Arch. f. Augenheilk., vol. 28, pp. 176—192, 1894.
5. Bauer, H., ‘v. Graefe’s Arch. f. Ophth.,’ vol. 42, p. 3, 1896.
6. Lepat, ‘Ann. d’Ocul.,’ vol. 101, 1889.
7. Niesnamoff, ‘v. Graefe’s Arch. f. Ophth.,’ vol. 42, p. 4, 1896.
8 Starling, ‘Journ. of Physiol.,’ vol. 19, 1896, p. 312.
9. Gronholm, ‘y. Graefe’s Arch. f. Ophth.,’ vol. 49, 1900.
dll
On the Filtration of Crystalloids and Colloids through Gelatine :
with special reference to the behaviour of Hamolysins.
By J. A. Craw, British Medical Association Research Scholar.
(Communicated by Leonard Hill, M.B., F.R.S. Received December 1, 1905,—
Read February 1, 1906.)
The current controversy between Ehrlich (1898, 1903) and Arrhenius and
Madsen (1902, 1904) on the physical chemistry of the neutralisation of toxins
by their specific antitoxins led the author (IV, 1905) to an investigation of
the relations existing between the toxin for red blood corpuscles secreted by
B. megatherium and its specific antiserum. One of the methods adopted
consisted in the filtration of mixtures of megatheriuin hemolysin and antilysin
through gelatine. The lysin was found to pass into and even through the
gelatine, whereas the antilysin was retained, and by means of the delicate
blood test for free lysin it was possible to demonstrate that the two
substances, on mixing in any proportions, do not completely neutralise each
other. These observations indicated that Ehrlich’s views on the toxin-
antitoxin reaction required considerable modification, but a closer investigation
showed that the hypcthesis advanced by Arrhenius and Madsen agreed even
less with the experimental facts. On the other hand, the results were in entire
harmony with the views advanced by Landsteiner (1903) and Bordet (1903),
which have been supported by Nernst (1904) and Craw (1905) that the toxin
is adsorbed by the antitoxin much as a dye is by a tissue. As this conclusion
may considerably modify ‘current ideas on the nature of the reaction and the
constitution of toxins in general, it seemed advisable to inquire further into
the physical chemistry of filtration through gelatine.
The present communication contains data of the filtration of various
erystalloidal and colloidal solutions, including megatherium lysin, through
various percentages of gelatine, under constant and variable pressures.
The work was partly carried out as Research Student at the Lister Institute
of Preventive Medicine and was completed at the London Hospital Medical
College by the aid of a scholarship from the British Medical Association.
Previous Work on the Gelatine Filter—The gelatine filter was introduced _
by C. J. Martin (1896), and consists of a Pasteur-Chamberland candle, the
pores of which are filled with solid gelatine. The filter is fitted into
a gunmetal jacket or filter case, which serves to hold the liquid to be filtered,
and the upper end of the closed filter case is connected with a supply of air
at a pressure of 30 to 100 atmospheres, which is used to force the liquid
VOL. LXXVII.—B. : 2A
312 Mr. J. A. Craw. On the Filtration of [ Dec. 1,
through the gelatine. From his observations with “wet” gelatine filters,
a.¢., filters containing gelatine from which the normal content of water had
not been removed by drying in air or otherwise, Martin* concluded that
gelatine was impermeable to colloidal substances such as globulin, albumin,
glycogen, and soluble starch, but partially permeable to albumoses and
dextrin, and completely permeable to solutions of crystalloids, eg., urea and
dextrose. It seemed, then, as if the gelatine filter was an instrument
destined to play an important part in the investigation of physiological
fluids.
Martin and Cherry (1898) applied the filter to the investigation of the
course of the reaction occurring between diphtheria toxin and antitoxin, and
likewise to the reaction of snake venom with antivenene, the toxin and venom
being filter-passers, whereas the anti-bodiest+ were retained.
From these experiments it seemed as if the toxin was completely neutralised
by the antitoxin, but further investigation of the mechanism of gelatine
filtration shows that no such absolute conclusion can be drawn. E. Waymouth
Reid (1901) showed conclusively that crystalloids do not pass “wet”
gelatine filters in unaltered concentration, and that although filters which
had been dried to constant weight in dry air allowed certain crystalloidal
solutions to pass unchanged, yet dextrose and sodium oleatet were partially
retained. Further, he found that the filtrate from serum} had not the same
composition as the proteid-free serum, and that the residual fluid left in the
filter case had a much higher concentration of organic substances of non-
proteid character than either the original serum or the filtrate. The Martin
filter is not, therefore, a simple means of separating crystalloids from colloids.
One must not, however, under-estimate its value as an instrument for the
analysis of physiological fluids, for although the filter shows considerable
differences in permeability to various ecrystalloidal substances, I find (IV,
1905) that these and inferior colloids are, on the whole, retained to a
small extent compared with typical colloids. The partial retention of
filter-passers has an important bearing on the conclusions to be drawn from
gelatine filtrations, for, if the concentration of the filter-passer to be tested
for be small in the original fluid introduced into the filter, the gelatine may
retain practically the whole amount and the filtrate contain only a quantity
below the experimental error of observation. This was found to be the case
for neutral mixtures of megatherium lysin and antilysin, and mixtures
* Loe. cit.
+ Of. also Brodie (1897) (1900).
{ Krafft (1902) considers soap solutions, such as‘sodium oleate, to be colloidal.
§ Cf. also Starling (1899).
1905. | Crystalloids and Colloids through Gelatine. 313
containing excess of antilysin, by the author;* the filtrates showed no
hemolytic power, whereas the gelatine had stored up a considerable amount
of free megatherium lysin. In this light Martin and Cherry’s observations
on diphtheria toxin and snake venom, mentioned above, are in entire agree-
ment with my results for megatherium lysin, the free toxin and venom of
the neutral mixtures being probably stored up in the gelatine of their filters.
On “ Wet” and “Dry” Gelatine Filters—Martin and others used the
filters in the “wet” state, zc. shortly after solidification of the gelatine
in the pores of the candle, and, therefore, containing a considerable quantity
of water, part of which on filtration will pass into the filtrate and so dilute
the substance filtered. To get rid of this difficulty, E. W. Reid removed
part of the water by drying Martin candles to constant weight in a current
of dry air. It seemed to me, however, that by drying the gelatine another
difficulty might be introduced, viz., a change in the size of the pores, which
would render observations with “dry” filters of doubtful value.
Method.—The rate of filtration of water through freshly prepared wet
filters was observed during one hour and compared with the rates during a
similar period, of filters which had been partially dried by standing in dry
air for 10 hours, 24 hours, and 3 days.
Rates of Filtration—The freshly prepared wet filters gave fairly uniform
rates, and allowed from 0°5 to 2 c.c. of water to pass per minute at
100 atmospheres pressure, the concentration of the gelatine in the pores
being 9 per cent. and the temperature 10° to 15° C. Under the same
conditions the partially dried filters allowed from 5 to 20 ec. to pass per
minute at the beginning of the filtration, but the rate rapidly decreased.
On reducing the applied pressure to about one atmosphere, and allowing
the water to flow through a partially dried filter at the rate of about
1 to 2 c.c. per minute, the filter gradually tightened, so much that after
the passage of 30 to 50 cc. a pressure of 100 atmospheres was necessary to
maintain the same rate. The longer the filters were dried the more marked
was the porosity and the greater the amount of fluid which had to be pressed
through before the filter tightened.
Conclusions.—On partially drying gelatine filters the gelatine shrinks,
and air passages are produced of greater diameter than the water passages
of a wet filter. The wide dry filter passages at first offer a free passage to
the fluid filtered, and no material change is to be expected in the percentage
composition of the latter. The filter gradually changes in character until
ultimately, a wet filter is obtained with pores of similar dimensions to those
of a freshly prepared wet filter. The various fractions of filtrate from a
* Loe. ett.
ey IN
314 Mr. J. A. Craw. On the Filtration of [ Dec. 1,
“dried” filter are not subject to the same conditions of filtration and are
not, therefore, comparable. Wet filters, on the other hand, show a much
greater constancy in rate of filtration, and are, therefore, to be preferred.
On the Preparation of the Fulters—To overcome the difficulty of dilution
of the filtrates by the water of wet filters, the gelatine to be used in filling
the candles was in several cases dissolved in the fluid to be filtered.
Thus the filtration of 0°8 per cent. sodium chloride and of megatherium
lysin took place through 9 per cent. gelatine which had been dissolved in
08 per cent. sodium chloride, and 1:54 per cent. potassium iodide was
filtered through 11 per cent. gelatine in 1°54 per cent. iodide. A Pasteur-
Chamberland candle of size B, which had in the majority of cases been
heated for 10 hours at least in a muffle furnace to remove organic matter,
was fixed into a brass socket with “Faraday” cement and fitted con-
centrically into the internally tinned gunmetal jacket or “filter case.”
After thoroughly washing through with about 400 cc. of hot water and
250 cc. of the solution of French gelatine to be used in forming the filter,
at a temperature of 37° C., the gelatine at 30° C. was slowly passed through
the cooling filter at an air pressure just sufficient to cause about 1°5 c.c. or
15 drops to filter per minute. The filtration was continued until no trace
of air bubbles was visible in the drops, after which a further 50 c.c. were
allowed to pass. During the final filtration the upper surface of the gelatine
solution in the filter case was not allowed to descend below a level 3 cm.
above the crown of the candle. The filter was then removed, drained,
placed in the neck of a flask containing a little water, and kept in the ice
chest at 6° to 8° C. until required. If the filter so prepared had an
obviously thick skin of gelatine upon its surface, this was removed by
immersing in the gelatine solution at 30° C. and draining as before. In the
comparative experiments with different concentrations of gelatine, the filters
were treated in the manner last described to get membranes of approxi-
mately equal thickness. Only those filters were used which allowed less
than 2 ¢.c. or about 20 drops per minute to pass at 100 atmospheres pressure
and 10° to 15° C., and the majority of filters allowed only about 0°5 c.c. per
minute to pass under these conditions. After use the candles could be
washed out with water at 50° C., dried, heated in a muffle furnace, and on
refilling with gelatine again gave reliable filters.
Filtration of a Typical Crystalloid, Sodium Chloride, through 9 per cent.
Gelatine.
Sodium chloride was chosen on account of its characteristic crystalloidal
properties and the important réle it plays in physiological fluids. Further,
1905. | Crystalloids and Colloids through Gelatine. 315
in a previous communication,* it was found that the filtrates from
megatherium lysin were strongly hemolytic, and it was necessary to
determine if this hemolysis was due in any degree to change in the
concentration of sodium chloride.
Method.—The filters used contained 9 per cent. gelatine in 0°8 per cent.
NaCl, and about 50 ec. of 0°8 per cent. NaCl were pressed through at
100 atmospheres and 10° to 12° C., the filtrates being collected in fractions
of about 4 ec. The NaCl content of 1 cc. of the fractions of filtrate, the
residual liquid left in the filter case and the original fluid, was determined
by titration with 1/100 normal silver nitrate, using potassium chromate as
an indicator. The hemolytic powers of the same fluids were determined
in these and all other experiments in this paper, unless otherwise stated,
by mixing 1 cc. of the respective fractions with 2 cc. of 2°5 per cent.
euinea-pig’s red corpuscles which had been washed and suspended in
0°8 per cent. NaCl. The mixtures were heated to 37° C. for three hours,
well shaken every 30 minutes, and allowed to sediment generally about
12 hours in the ice chest at 6° to 8° C.
The intensity of tint of the supernatant fluid was then determined by
comparison with the scale of a von Fleischl hemoglobinometer which had
been standardised by a blood solution of known content. Complete
heemolysis is indicated by the index (100).
Hiaminatiion for Sodiwm Chloride and Hemolytic Power.—Experiment
No. 2, Table I, represents one out of four similar experiments.
Original Fluid: 0°81 per cent. NaCl given in the table as Orig. (100).
Filtrates: The relative concentrations of the Ist, 4th, and 14th fractions
were (47'1), (89°5), and (99-2) respectively.
Residual Fluid: When tested immediately after decompression had the
value (100) exactly, but, on allowing to stand 12 hours in the filter case,
showed the relative concentration (105).
Hemolytic Powers: No hemolysis was obtained under the standard
conditions with either filtrates, residual fluid, or the original saline.
Conclusions—The typical crystalloid, sodium chloride, is markedly
retained on filtration through gelatine which originally contained the same
concentration of saline as the fluid filtered. The salt taken up by the
gelatine is expressed or diffuses into the residual fluid after decompression.
The diminution in salt concentration in the filtrate is insufficient to cause
hemolysis under the standard conditions.
* Loe. cit.
316
Mr. J. A. Craw. On the Filtration of
[Dee. 1,
Continued Filtration of Sodiwm Chloride through 9 per cent. Grelatine.
As the last fraction of filtrate in No. 2 did not quite reach (100), it seemed
desirable to ascertain whether on continued filtration this value could be
obtained.
Method.—The residual fluid of No. 2, which had stood in the filter case
Table I.
Sodium chloride, per cent. | Megatherium lysin, H.Is.
No. ... 1. 2. 2A | 2B. | 3 4 5 6 le | 8
1Es © 0°88. 0°81. | HBu. | 15 11. 9. 7°5 8.
23 °9 47 “1 101 °5 | 97°4.| 16-7 | 0:21 0-7 | 0°5| 0-0) 0-5 Oro
5 31 °4 55 ‘1 103°3 | 97°8 | 41°7.| 5:8) 05) 00; O00} 50| 3°5
8 39 0 76°1 99°6 | 96:0 | 47°8 | 60-4) O00 |] 2:3] 0:0)17-7 10-7
£ | 49:0 | 89°5 | 101-1 | 93:1] 56:9] 74:0] 0:0 | 5:1] 3-7 | 25-0 | 178
=| 56 °0 95 °3 99°6 | 94:2 | 65°6 | 85:4] O'°0 | 5:1] 27°8 | 34°6 | 21°4
os 64 +1 96 °7 97°6 | 93°5 | 70°6 | 88:5 | 0:0] 7:1 | 46°3 | 45:1 | 25°0
a 71:1 97 6 100°7 | 95:7 | 76:8 | 88:5 | 0:0} 8:6} 33°9 | 48°8 | 28°5
& 761 97 °6 97°6 | 94:2 | 80:4] 90'°6| 00) 8°6| 47°8 | 48°8 | 38°5
3 79 °2 97 6 = 94:2 | 88:1 | 90°6 | 0:0 | 8°6 | 47:0 | 48°8 | 32:1
& 81:7 99 -2 — 96:0 | 85:0 | 90°6 | 0:0 | 12°38 | 47-0 | 48-4 | 32:1
a 83 °3 99 °6 —_— 94°9 | 86°3 | 90°6 | 0O°0 | 12°8 | 45°6 | 48°4 | 35-7
i 112°6 | 100-0 —_ 96°8 | — _ 2°3 | 8-0 | 47:0 | 49°3 | 35°7
im 107 ‘8 99 -6 — 96°4 | — — 3°7 | 10:0 | 46°3 | 48°8 | 50-0
8 107 ‘8 99 °2 _ 96°4 | — — |100; — — | 48-4 | 50°0
a 98-4 = — | 96-4 —}|—}]—|— | — | 50:0
96 -2 — — — — — —_— _— — — | 50°0
93 -7
1384°9 | 100:0 | 100°0 |100°0 110 0 |125-0 | 85:0 | 62:9 | 55°6 | 52-7 | 67°8
Res. :
C.c. c.c. C.c. cle.) eke: c.c c.c, | ce C.c c.c
20 44, 65 40 45 _ AT 45 34 31
100‘0 | 100°0 | 100°0 /100-0 |100°0 |100°0 | 50°0 | 50°0 | 50°0 | 50°0 | 50°0
Orig: C.c. C.c. C.c. C.c. | ¢.c. Ca | G@ | Ga | aa | Ge.
100 112 112 120 105 = 110 105 100 110 105
overnight, was poured out, and the filter case refilled with 112 cc. of saline,
of which 40 c.c. in 5 cc. fractions were pressed through.
Hxamination for Sodium Chloride—Experiment No. 24, Table I.
Original Fluid: 0°81 per cent. NaCl = Orig. (100).
Filtrates: The 1st and 2nd fractions contained (101°5) and (103°3) respec-
tively, and those succeeding showed values varying about (100).
1905. | Crystalloids and Colloids through Grelatine. 317
Residual Fluid: Titrated immediately after decompression (100), after
12 hours in the filter case (110).
Conclusion—On renewing filtration the filtrate may contain a higher
percentage of salt than the original fluid, due probably to the temporary
decompression, for on continued recompression the concentration tends to
become less than (100).
Continued Filtration of Sodium Chioride with Megatherium Lysin and
Antilysin through 9 per cent. Gelatine.
In a former paper,* mixtures of lysin and antilysin were pressed
through gelatine which had been tested for tightness by the filtration of
saline. ‘he redistribution of salt under these circumstances was now
determined.
Method.—A filter through which 10 c.c. of 0°81 per cent. NaCl had been
pressed, the original gelatine being 9 per cent. in 0°81 per cent. NaCl, was
decompressed, the residual salt solution removed, and the filter case refilled
with 120 c@c. of a nearly neutral mixture of equal volumes of a fluid
megatherium lysin and 5 per cent. antilysin in saline, of which 75 ce. in 5 c.c.
fractions were pressed through.
Hzamination for Sodium Chloride—Experiment No. 28, Table I, is one of
two practically identical filtrations.
Original Fluid: Contained 0°81 per cent. NaCl = (100). Hemolytic
Index (16°8).
Filtrates: As in No. 2A the 1st and 2nd fractions were the most con-
centrated in NaCl, but they did not reach (100), and the final fraction was
(96:4). The average hemolytic index of the whole filtrate was (0°5).
Residual Fluid: The results were entirely similar to those of No. 2a as
regards NaCl. Hemolytic Index (38'6).
Conclusion —tThe redistribution of salt is insufficient to give a trace of
hemolysis under standard conditions.
Temporary decompression even for a few minutes allows a higher per-
centage of salt to pass into the filtrate than would be obtained by constant
pressure.
Filtration of Sodiwm Chloride with Butyric Acid through 9 per cent.
; Gelatine.
Butyric acid in saline acts as a very strdng heemolysin, and, as it possesses
erystalloidal properties, it appeared to be of interest to compare its filtration
phenomena with those of megatherium lysin in saline. Further, butyric
* Loe. cit.
318 Mr. J. A. Craw. On the Filtration of [ Dee. 1,
acid greatly diminishes the surface tension of saline against air, and the
possibility of a change in the surface forces between saline and gelatine formed
a second point of interest, which is discussed towards the end of this paper.
Method—A_ solution of 0°81 per cent. NaCl containing 48 per cent.
butyric acid was pressed through 9 per cent. gelatine in 0°81 per cent. NaCl
at 100 atmospheres. The content of the fractions of filtrate, etc., in NaCl
was determined as before, and the butyric acid was estimated by titration
with standard sodium hydrate, using phenol-phthalein as indicator. The
hemolytic powers of the various fluids were also determined by the time
taken for 1 c.c. of the fractions to hemolyse completely 2 cc. of a 2°5 per
cent. suspension of guinea-pig’s corpuscles in 0°8 per cent. NaCl at 16° C.
Hxamination for Sodiwm Chloride—HExperiment No. 3, Table I The
redistribution of salt was qualitatively similar to that which had been
obtained in the absence of butyric acid. The amount passing into the filtrate
was, however, smaller.
Examination for Butyric Acid and Hemolytic Power.—Original: 48 per
cent. butyric acid = (100) HBu. Heemolytic time = 8 minutes.
Filtrates: The 1st and 2nd fractions contained (0°2) and (58) HBu
respectively. The 1st fraction agglutinated the test blood strongly, and the
2nd hemolysed completely in 50 minutes. The 8th to the ilth fractions
contained (90°6) HBu, and the hemolytic time was 12 minutes.
Residual Fluid: Concentration (125) HBu. Hemolytic time, 5 minutes.
Conclusions.—Butyric acid is retained by gelatine to a considerable extent.
The gelatine appears to retain more sodium chloride in the presence of
butyric acid.
Liltration of Sodiwm Chloride through 9 per cent. Formalised Celatine.
It seemed probable, from the marked change which takes place in gelatine
on being exposed to formic aldehyde, that such gelatine would show a different
permeability to that already found for ordinary gelatine. On the other hand,
the possibility of investigating physiological fluids in an apparatus which
could be thoroughly and easily sterilised by means of “formalin” might
recommend the use of formalised gelatine filters.
Method.—A filter containing 9 per cent. gelatine in 0°8 per cent. NaCl was
fitted into the filter case, and the latter filled with a solution of 0°8 per cent
NaCl containing 10 per cent. of commercial “ Formalin.” A few cubic centi-
metres of fluid were pressed through, and the remainder allowed to stand in
the filter case overnight. The solution was then removed, and, after about
12 hours, 100 ec. of 0°88 per cent. NaCl were placed in the filter case, and
pressed through in fractions of 4 to 5 c.c.
1905. | Crystalloids and Colloids through Gelatine. 319
Lxamination for Sodium Chloride.—Experiment No. 1, Table I.
Original Fiuid: 0°88 per cent. NaCl represented as Orig. (100).
Filtrates: No formaldehyde or formic acid could be detected. From the
1st to the 11th fraction the NaCl gradually increased from (23:9) to (83°3),
at 100 atmospheres filtration pressure. At this point the pressure was
removed and filtration resumed after an interval of eight hours. Six fractions
of 5 cc. each were then removed, and gave values decreasing from (112°6)
to (93-7).
Residual Fluid: Tested immediately after the last decompression con-
tained (1349).
Conclusions.—Formalised gelatine containing sodium chloride retains
sodium chloride from a solution, on filtration, to a greater extent than
ordinary gelatine. The decompression in this case also leads to the
immediately following filtrates having a higher concentration of salt than
the original fluid, but the effect is more marked than with ordinary gelatine.
The concentration of the residual fluid is also much greater than with
ordinary gelatine.
Filtration af Megatherium Lysin Through Various Percentages of Gelatine.
Megatherium lysin diffuses slowly compared with crystalloids in general,*
and is probably of semi-colloidal character. It seemed probable that a
substance of this type would be considerably more affected by a change in
the concentration of gelatine than a good filter-passer such as sodium
chloride. Hardy’s (1899) work on gelatine pointed also to the possibility of
considerable change in the structure of the jelly at about 7 per cent. gelatine,
which might lead to markedly different degrees of permeability between 7
and 15 per cent gelatine filters.
Method.—Filters containing 15, 11, 9 and 7°5 per cent. gelatine in 0°8 per
cent. NaCl were tested for tightness with 0°8 per cent. NaCl, of which
10 ¢.c. were pressed through in each case to ensure a concentration of NaCl
in the succeeding filtrates of about 0°8 per cent. The fluid examined was
the hemolytic filtrate from a broth culture of B. megatherium diluted with
an equal volume of 0°8 per cent. NaCl.
Filtration was carried through at a pressure of 100 atmospheres and a
temperature of 10° to 12°C. and the filtrate was ‘collected in fractions of
about 4 ce.
Hxamination for Lysin—Table I.
Original Fluid—The hemolytic index (see above) was (50).
* Cf. Craw (IV, 1905).
320 Mr. J. A. Craw. On the Filtration of [Dee, 1,
Filtrates: Experiments Nos. 4, 5,6 and 7, Table I, show the hemolytic
values of the succeeding fractions through 15, 11, 9 and 7-5 per cent.
gelatine respectively. The average indices for the whole filtrates were in the
order given above (1°5), (6:9), (31°7), and (36°9).
In Experiment No. 8, Table I, 50 cc. of 20 per cent. horse serum in
0:8 per cent. NaCl had previously been passed through a 75 per cent.
gelatine filter. In this case the permeability to lysin was slightly less than
that of a similar filter without serum, the average indices of ‘the filtrates
being (30°6) and (369) respectively.
Gelatine: On melting out the gelatine, after filtration, at 37° C., and
mixing with the test blood, the latter was in all cases rapidly and completely
heemolysed, index (100). Control experiments showed that saline on being
pressed through gelatine which had been used to filter lysin became strongly
hemolytic. The original gelatine had no hemolytic effect in the standard
time.
Residual Fluids: The residual fluids in all cases showed average indices.
which were higher than that of the original fluid. The lowest portions of
residual fluid had generally higher indices than the portions towards the
upper surface. With rising concentration of gelatine the hemolytic power
increased, thus with 7°5 per cent. the residue had an index (52°7) whereas.
with 15 per cent. it was (85).
Conclusions—Megatherium lysin is retained to a greater extent than
sodium chloride, and more is retained with higher concentrations of gelatine.
The residual fluids have higher concentrations than the original and the
concentration is greater with higher percentages of gelatine. Diffusion and
expression of lysin from the gelatine into the residual fluid are insufficient to
account for the increased concentration immediately after decompression, and |
it seems as if the water of the original fluid could pass into the gelatine
more readily than the lysin.
It seems possible, under the conditions of preparation, that during the
draining of the filters from the various percentages of gelatine at 30° C., the
filters with the higher concentrations would retain thicker surface layers of
gelatine and so exaggerate the differences in permeability.
Filtration of a Typical Colloid, Ferric Hydrate, through 11 per cent. Gelatine
‘ under Varying Pressure.
As ferric hydrate in colloidal solution shows no appreciable tendency to
diffuse, any redistribution of the colloid by the gelatine filter must be
explained on some other basis than that of diffusion.
Method.—A_ 5-per-cent. solution of colloidal ferric hydrate was prepared
1905. | Crystalloids and Colloids through G'elatine. 321
by adding ammonium carbonate to ferric chloride and dialysing for three
weeks; it gave no trace of red coloration with potassium thiocyanate, but
showed the characteristic reaction of colloidal ferric hydrate, viz., a slight
yellowish precipitate. The filter used contained 11 per cent. gelatine which
had been dialysed for 24 hours. The content of the fractions of filtrate, etc.,
in iron, was determined by converting the hydrate into chloride and
estimating colorimetrically with potassium thiocyanate.
Table II.
Colloidal ferric hydrate. Neutral red. Iodine in potassium iodide.
Rollin | Pres- Per- sail ae Pres- Per- Wola Pres- Per- Per-
ne sure in | centage aie sure in |centage Ot sure in | centage} centage
; atmos. Fe. ne atmos. | N.R. “| atmos. I,. KI.
4 100 0:0 4 100 0:0 1 100 5 68 ‘8
f 4 100 0-7 4 100 2°3 4 100 8 69 °9
g 4 100 06 4 100 0:04] 4 100 17 70°9
£ 4 100 0-4 3 20 | 0:03] 4 100 | 19 69 9
P| 4 100 0-4 10 100 0°30) 4 100 21 69 ‘9
a 4 100 0-4 10 100 0-78 | 4 100 23 69 ‘9
S A 100 O 10 100 0:90] 4 100 25 68 ‘3
g 4 100 0:0 10 100 Waly |} al 15 20 69 ‘9
B 4 100 0-0 10 100 3°00 | 5 100 33 71°5
S 1 100 0:0 10 100 3:20 | 5 100 33 73°0 |
eS 05 20 | 250-0 20 100 4°68 | 4 100 35 72:0
ry 1 100 4°75 = = = 0°75 (0) 10) 43 °O |
a 2 100 0:9 — — — 0°75 | 100 25 93-5 |
= 3 50 0-7 _ —- | = 6 100 37 90 °5 |
an 05 15 66 6 — —- | — 6 100 50 86 ‘0
0°75 50 1:0 — — — il 0) 1 34 °4,
0:25 30 0% — — — 5 10 25 94 6
0°5 20 0°3 ~— — — 2 100 30 92 °5
0°75 10 O-1 — — — 3 50 30 96 °8 |
0°25 50 0-2 — — 4 100 30 91 °4 |
1 100 0:2 — — 5 50 30 | 85:0
5 15 0-4 — — — 5 25 30 | 82°8
4 100 0°4 = = = 5 25 25 | 79°5
0°5 15 0 “4 — — — 5 25 Py |) al |
4 100 0-4 |
Res. | 50 == ))))/5180/-08. |) 20 — | 18-75 | 35 | eof teen |
|
|
Orig.| 120 — 100 ‘0 120 — |100°0 {120 — | 100 | 100 ‘0
| |
Examination for Ferric Hydrate-—Table II gives the relations obtained in
one out of three similar experiments.
Original Fluid: Five per cent. ferric hydrate = (100) Fe.
Filtrates: The 1st and 2nd fractions contained (0:0) and (0°7) respectively,
and in the succeeding fractions the amount diminished until in the 8th no
322 Mr. J. A. Craw. On the Filtration of [Dec. 1,
trace could be found at 100 atmospheres filtration pressure. On suddenly
diminishing the pressure to 20 atmospheres the liquid percolating through
was very intensely coloured and gave the value (250). Re-establishing the
pressure of 100 atmospheres, the filtrates became less and less intensely
coloured (4:75) and (09). A drop in the pressure to 50 atmospheres did not
materially change the concentration, but a further drop to 15 atmospheres
gave a filtrate with the value (666). Increasing the pressure to 50
atmospheres caused the filter to tighten once more with respect to the
hydrate, and on gradually decreasing the pressure to 10 atmospheres the
filter remained tight. In this condition sudden variations of pressure from
100 to 15 atmospheres had but a slight effect on the permeability of the
filter.
Residual Fluid: The colour was more intense than that of the original
fluid and its content was (180) Fe.
Conclusions—The gelatine fifter is slightly permeable to the typical
colloid ferric hydrate, but at constant pressure the permeability decreases as
the filtration proceeds. The permeability is increased enormously by
suddenly diminishing the pressure, but is not much affected by a gradual
diminution. After gradual diminution of pressure a filter is obtained which
does not markedly change in permeability on suddenly varying the pressure.
The high concentration of the residual fluid is probably due to the water
penetrating the gelatine easily, whereas the ferric hydrate is largely left on
the surface of the gelatine, where it forms a concentrated solution of higher
specific gravity than the rest of the fluid and so gives rise to convection
currents which cause it partly to mix with the remainder of the fluid in the
filter case. The candle retains a skin of colloidal ferric hydrate, and the filter
most probably at the beginning of the filtration acted as a simple
gelatine filter, but subsequently as a compound ferric hydrate gelatine filter.
Filtration of a Staining Colloid, Neutrai Red, through 11 per cent. Gelatine
under Varying Pressure.
Preliminary experiments with horse serum and solubie starch showed
qualitatively similar effects to those obtained with colloidal ferric hydrate,
and it seemed probable that the majority of colloidal solutions would behave
in a similar way on filtration through gelatine.
It seemed probable, however, that those colloidal solutions which atia
gelatine would show considerable difference in behaviour.
Method.—A 0°5-per-cent. solution of neutral red in distilled water was
pressed through 11 per cent. dialysed gelatine and the content of the fractions
of filtrate, etc., determined colorimetrically.
1905. ] Crystalloids and Colloids through Gelatine. 323
EHzamination for Neutral Red.—Table II.
Original Fluid: 0°5 per cent. neutral red = (100) N.R.
Filtrates: The 1st, 2nd and 3rd fractions showed (0:0), (2°3) and (0:04)
respectively. A sudden diminution in pressure from 100 to 20 atmospheres
caused a slight decrease in the value of the filtrate (0°03). On raising the
pressure once more to 100 atmospheres, the succeeding fractions gradually
increased in content up to (4°68) N.R. ,
Residual Fluid: The colour intensity was much diminished and indicated
only (18°75) N.R.
Conclusions.—The gelatine filter is slightly permeable at the beginning of
filtration to neutral red, but the permeability decreases, as in the case of
colloidal ferric hydrate, on continued filtration at constant pressure.
Diminution of pressure has the opposite effect to that obtained with sodium
chloride and ferric hydrate as the permeability tends to decrease. The filter
gradually becomes stained throughout and the permeability increases
correspondingly. —
Filtration of a Staining Crystalloid, Iodine in Potassium Iodide, through
11 per cent. Gelatine, under Varying Pressure.
Iodine was chosen as a crystalloidal substance having the property of
staining gelatine, and potassium iodide was used as its solvent in water.
The interest of the experiment centred chiefly in the relative behaviour of the
two substances and the mutual influence they exert on each other during
filtration under varying pressure.
Method.-—A solution of 0:214 per cent. iodine in 1°54 per cent. potassium
iodide was filtered through 11 per cent. gelatine in 1°54 per cent. potassium
iodide. The content of the fractions of filtrate, etc., was determined by
titration with sodium thiosulphate, using starch as an indicator for the iodine
and with silver nitrate, using potassium chromate as indicator for the
potassium iodide.
Examination for Iodine and Potassium Iodide.—Table II.
Original Fluid: 0:214 per cent. Iz = (100) Iy. 1:54 per cent. KI = (100) KI.
Filtrates: The 1st c.c. contained a considerable percentage of iodine (5)
and likewise of potassium iodide (68°8). On continued filtration at constant
pressure the iodine concentration rose steadily to (25); the iodide rose slightly
and then fell to (68°3). On suddenly diminishing the pressure from 100 to
15 atmospheres the iodine concentration diminished and the iodide increased.
On raising to 100 atmospheres the iodine rose, whereas the iodide rose and
then fell in concentration. Removing the pressure for 12 hours the few drops
of liquid which percolated through contained no iodine and only (43) of
324 Mr. J. A. Craw. On the Filtration oy [Dec. 1
iodide. Re-establishing the pressure of 100 atmospheres the iodine content
rapidly increased, whereas the iodide in the first few drops was abnormally
high (93°5), but decreased in the second fraction of 6 c.c. to (860). Once
more removing the pressure for three hours the iodine diminished to (1:0)
-and the iodide to (34'4), confirming the former result. The pressure was now
slightly raised, viz., to 10 atmospheres, when the iodine became (25) and the
iodide reached the highest value so far (946). A further rise in pressure to
100 atmospheres increased the iodine to (30) and diminished the iodide to
(92°5). Variation between 100 and 50 atmospheres did not further affect the
‘iodine content but did influence the iodide. Finally,a gradual fall in pressure
ito 25 atmospheres caused both iodine and iodide to diminish in concentration.
Residual Fluid: The concentration of iodine was only (30), whereas the
iodide was (109°7).
Conelusions—The gelatine absorbs a large amount of iodine, but is easily
permeable to the same and the permeability rises and falls, as in the case of
meutral red, with rising and falling pressure. The increasing concentration of
iodine in the gelatine increases the power of the latter in retaining potassium
iodide. On entirely removing the applied pressure the gelatine robs the
liquid percolating through almost entirely of its iodine and the iodised
‘gelatine abstracts about two-thirds of the iodide.
The filtrates, after a drop in the pressure, show an increased amount of
iodide on recompression similar to the effect observed in the filtration of
-sodium chloride.
On the Influence of the Nature of the Solution and of Varying Pressure on the
Rate of Filtration.
A few preliminary measurements of the rates of filtration of various
-solutions were made to elucidate some of the physical properties governing
the action of the gelatine during filtration.
Method.-As a rough approximation the number of drops per minute
falling from the nozzle of the filter case was taken as a measure of the rate
.of filtration. The rate was determined after the first 2 c.c. had passed, and
again after about 50 cc. in all had been collected.
Rates of Filtration—Table III shows that distilled water, 0-8 per cent.
sodium chloride and megatherium lysin pass 9 per cent. gelatine in the initial
stage more rapidly than in the final under constant pressure. Solutions
containing 0:214 per cent. iodine, with 1°54 per cent. potassium iodide, and
5 per cent. colloidal ferric hydrate pass 11 per cent. gelatine with similar
~variations in rate. For all these solutions the decrement in rate is practically
1905. | Crystalloids and Colloids through Gelatine. 325
the same. On filtering horse. serum in full strength, however, the decrement
in rate was found to be abnormally large.
Table III.
4 KI, | NaCl.| NaCl | NaCl NaCl | Dist. Coll.
pepiaunios. 120 C-1) NaCl, I. | lysin. | toluol.| Am.Alc.| HBua. | water. | Fe(HO);. SST
Initial drops per| 7 | wl 8 16 18 6 9 6 6
minute
Final drops per 4 4, 5 18 60 18 4 4 1
minute
A solution of 0°8 per cent. saline containing toluol as a fine emulsion did
not show any decrement.
On the other hand, 0°8 per cent. saline containing either amylic alcohol or
butyric acid (48 per cent.) gave very distinct increments in the rate of
filtration.
Table IV gives a comparison of the rates obtained for distilled water and 0°81
per cent, sodium chloride containing 4°8 per cent. of butyric acid. It is evident
that distilled water filters more and more slowly, whereas the saline butyric
acid passes more and more rapidly. The effect of removing the pressure for
two minutes enables the distilled water on re-establishing the pressure to pass
through at a higher rate, and if the pressure be removed for 10 minutes the
gelatine regains completely its original rate of filtration.
Table IV.
Distilled water. Sodium chloride and butyric acid.
Pressure | Time in | Drops per| C.c. per | Pressure | Time in | Drops per | C.c. per
in atmos. | minutes. | minute. minute. | in atmos. | minutes. minute. minute.
100 0 7 —_ 100 ¢) 6 =
100 13 6 0°5 100 2 5 =
100 21 5 0°5 100 5 9°5 =
0 23 (0) — 100 u 11 0-7
100 29 6°5 0°6 95 15 9°75 0-6
100 32 5 0°5 90 20 9 =
100 40 4:5 0°3 100 23 11 0-6
0 50 0 — 100 35 13 0-6
100 73 9 0-7 100 42 14 0-7
100 83 5°5 0°5 100 49 15 0-7
100 133 4°5 04 100 64 16 0°8
100 71 18 0:9
100 TA 18 aL Sit
326 Mr. J. A. Craw. On the Filtration of [ Dee. 1,
Conclusion.—Solutions of salts such as sodium chloride, or colloids such as
ferric hydrate, will not appreciably modify: the rate of filtration through
gelatine unless in concentrated solution. Amylic alcohol and butyric acid
accelerate the rate of filtration.
It seemed possible that this effect might be due to the condensation of
amylic alcohol and butyric acid on the walls of the gelatine pores, and that
the change in viscosity might account for the increased rate. This, however,
does not seem probable, for a toluene emulsion did not markedly affect the
rate of filtration, and as the viscosities of the toluene and amylic alcohol used
were found to be 0°49 and 4:4 respectively when compared with distilled
water as unity, one might conclude that the toluene would accelerate, and the
amylic alcohol retard, the rate of filtration.
On the other hand, the action of amylic alcohol and butyric acid is
consonant with the view that the surface forces between gelatine and saline
are modified. The surface tension of saline against air diminishes with
increasing concentration of both amylic alcohol and butyric acid. Both of
these substances markedly influence the rates of sedimentation of solid
suspensions through water,* and this fact seems to have a close connection
with their influence on the rate at which water passes through a porous solid
or jelly such as gelatine.
The effect of continued pressure on gelatine seems to be a tightening of the
pores, which is nearly complete at 100 atmospheres in 40 minutes, and
decompression apparently allows the pores to resume their original dimen-
sions in about 10 minutes at atmospheric pressure.
On the Expression of Water and Gelatine from a Wet Filter.
In agreement with E. W. Reid, the filtrates were found to contain gelatine,
but in variable amounts. Thus the first fraction of about 4 c.c. usually con-
tained most, and after 20 c.c. had passed the gelatine was only present in
traces. Rough estimations with tannic acid indicated about 0°5 per cent.
gelatine in the first, and less than 0:1 per cent. in the fifth fraction on filtering
distilled water. These contents of gelatine were not found to influence the
titrations given above.
It has been hitherto assumed that the water in the pores of a wet filter is
largely expressed before the liquid undergoing filtration appears in the filtrate
undiluted by the same. For this reason Martin} discarded the first 10 c.c. of
filtrate, and E. W. Reid} concluded that the concentration of even a second
fraction of 25 c.c. might be considerably reduced by this dilution. It seems
to me, however, that the effect of the water in the gelatine on the concen-
* Cf. Craw (1904). + Loe. cit. t Loc. cit.
1905. | Crystalloids and Colloids through Gelatine. 327
tration of the filtrate is neither so marked nor so long continued as has been
imagined, for the following reasons. In the first place, the filtrates from
saline through gelatine containing the same concentration of saline are at first
only about half as concentrated as the original saline. Secondly, on decom-
pression, and again filtering, the filtrate may contain a higher concentration
of salt than the original saline. This seems conclusively to show that much
of the dilution of the filtrate is due not to the water of the filter, but to the
retention of the salt by the gelatine. Further, Reid found the quantity of
water in freshly prepared filters to vary between 2 and 13 grammes—~.c., pre-
sumably, the amount which can be removed by dry air. It is probable that
only a smal) part of this water will be removed by the passage of a slow
current of liquid through the gelatine, the remainder being retained by
adsorption or inbibition forces, etc. In this connection an observation which
was made with all the filtrations given in this paper may not be unimportant.
It was found that the sum of the volumes of the filtrate fractions and residual
fluid in any one filtration experiment was less than the volume of the
original fluid introduced into the wetted apparatus. It is a well-known
fact that water can be forced into gelatine by. pressure, and as a large part of
the gelatine in the pores of the filter, during filtration at 100 atmospheres,
must be under considerable pressure, it seems reasonable to assume that part
of the original water of the wet filter is more firmly bound, and that about
5 ¢.c. are imbibed partly from the water of the filter and partly from the fluid
filtered. The rapid passage of iodine, neutral red, and colloidal ferric hydrate
into the filtrate also point to a considerable percentage of the first fractions
of filtrate being contributed by the fluid filtered. It seems, on the whole, as
if the free water of the filter is almost wholly removed in the first fraction of
5 cc. filtrate.
The Application of Mechanical and Adsorption Hypotheses to the Filtration
Phenomena of Gelatine.
Mechanical Hypotheses—The most obvious explanation of the retention of
colloidal substances by the gelatine filter is that the colloidal molecule or
grain is too large to pass through the pores. Martin* has advanced the view
that the non-filtration may be due either to the size of the molecules or to
some interaction between the colloidal membranes and the dissolved colloidal
molecules. It has, however, been shown by E. W. Reid (1904), Gatin-
Grazewska (1904), and others that proteids, glycogen, and other typically
colloidal substances, exert no measurable osmotic pressure in solution, do not
influence the freezing point of the aqueous medium, and in general diffuse
* Loe. cit.
VOL. LXXVII.—B. 2B
328 Mr. J. A. Craw. On the Filtration of [ Dee. 1,
very slowly, if at all. These facts lend much probability to Oswald’s view
that colloidal solutions may be regarded as very fine suspensions. As the
molecular weights of these substances are as yet quite indefinite, it would
seem more justifiable to speak of the colloidal “grain” than of the colloidal
“molecule.” Accepting this view of the nature of colloidal solutions, the
action of the filter admits of an apparently satisfactory explanation on a
purely mechanical basis.* During the filtration, for example, of colloidal
ferric hydrate, Table II, the first fraction contained none of the colloid, and
probably consisted chiefly of the water of the filter. In the succeeding
fractions a little ferric hydrate passed through the larger pores, but these were
speedily blocked by the particles. Under the pressure of 100 atmospheres
the pores are probably compressed, and the effective passages are soon com-
pletely blocked by ferric hydrate. On suddenly diminishing the pressure the
gelatine begins to return to its original condition, and the effective passages
increase in number and diameter. The condensed ferric hydrate, no longer
completely blocking the widened passages, is swept out of the gelatine by the
oncoming fluid, and so gives a filtrate with an abnormally high concen-
tration.
On the other hand, by gradually diminishing the pressure the ferric
hydrate has time to fill the new and the gradually-widening pores, so that
the filtrate contains only a trace of colloid. If the ferric hydrate be not
expressed on recompression, it is conceivable that the compound filter of
ferric hydrate and gelatine would be less affected as regards permeability by
variation of pressure than the original gelatine. Much as this view has to
recommend it, it does not seem to be the whole explanation, as it does not
satisfactorily account for the phenomena observed with sodium chloride. It
is highly improbable that sodium chloride is retained by a mechanical
blocking of the passages, and that it is swept in high concentration into
the filtrate, on decompression, because the condensed sodium chloride is no
longer of the same dimensions as the widened passages. Recourse must,
therefore, be had to some other view, which may considerably modify the
explanation given for colloidal substances.
Adsorption Hypotheses—Porous substances, powders, and fine suspensions
of solids in aqueous media have the power of removing salts and other
substances from solution. The action is attributed to forces of the same
nature as those which give rise to adhesion and the wetting of a solid by
a liquid, eg., of clean glass by water. The phenomenon usually called
adsorption is common to all substances with highly developed surfaces, and
* Of. also M. Traube (1866, 1867). Traube regards precipitation membranes as atomic
and molecular sieves.
1905. | Crystalloids and Colloids through Gelatine. 329
the intensity of the adsorption depends not only on the physical condition
but also on the chemical nature of the active surface. Gelatine being,
probably, a porous substance, presents a large surface to the enclosed fluid,
and it seems highly probable that when this fluid contains crystalloids or
colloids adsorption will take place. Thus, gelatine containing 0°8 per cent.
saline apparently retains about one-half of its content in sodium chloride,
and the retention of crystalloids and colloids on filtration through gelatine at
constant pressure seems to admit of explanation on the basis of adsorption.
The results obtained in the investigation of the adsorption phenomena of the
sedimentation of silts through aqueous solutions may therefore be applied to
what apparently is the converse of that process, viz., the passage of aqueous
solutions through porous solids or powders, under pressure. This is rendered
highly probable from the fact that the rate of sedimentation of a silt through
saline is generally accelerated. by the presence of butyric acid, amylic alcohol,
and other substances influencing surface tension, and similarly the rate of
filtration through the porous gelatine is accelerated by the same substances.
An additional factor may, however, come into play in gelatine filtration. The
effect of pressure on gelatine containing a solution of salt will be to cause the
absorption of water. It seems permissible to assume, in view of Hardy’s
work on the structure of gelatine, that this additional water will be taken
up by the web mass and will probably lead to a diminution in dimensions
of the pores or web spaces. This view would account for the continued
diminution in rate of filtration at constant pressure. The process should
show some similarity to the passage of a solution into filter paper, where the
water passes in more readily than the substance in solution.
A sudden diminution in pressure will lead to the rapid expulsion of the
imbibed fluid, which will sweep the adsorbed matter into the widening pores.
The gelatine itself will thus express part of this concentrated fluid, and at
low pressures highly concentrated filtrates will be obtained. This will take
place markedly when the adsorption is easily reversible, but the less reversible
adsorptions of certain staining substances, ¢.g., neutral red and iodine will not
give this effect. Further compression and decompression would presumably
lead to a more unitorm distribution of the adsorbed substance throughout the
gelatine, and as the filter is now also more saturated, the effect of variation of
pressure on the concentration of the filtrate would be less marked.
Summary of Conclusions.
1. Wet gelatine filters are to be preferred to those which have been
partially dried, as the former have more uniform rates of filtration, and dilu-
tion of the filtrate by the water of the gelatine can be largely eliminated.
2B 2
330 Mr. J. A. Craw. On the Filtration of [ Dec. 1,
2. Under constant pressure the gelatine of the filter absorbs water, and its
porosity gradually decreases ; on decompression this water is expressed, and
the original porosity is rapidly regained.
3. Gelatine of a definite concentration apparently has a specific permea-
bility for different crystalloids and colloids; the value is high but not
complete for the crystalloids sodium chloride, potassium iodide, and butyric
acid, and it is low, but not zero, for the colloids ferric hydrate, serum and
soluble starch. 7
4. As filtration proceeds the crystalloids pass through in increasing con-
centration, whereas the colloids rapidly decrease to zero.
5. The simultaneous filtration of two substances may influence their
specific permeabilities, thus butyric acid alters the permeability to sodium
chloride, and iodine that of potassinm iodide.
6. Variations in the gelatine influence the permeability, eg., formalised
gelatine is less permeable to sodium chloride than ordinary gelatine, and
15 per cent. gelatine is less permeable to megatherium lysin than 75
per cent.
7. Variation in the pressure causes remarkable changes in permeability.
A sudden diminution of pressure gives highly concentrated filtrates of both
erystalloids and colloids, whereas a gradual diminution has practically no
effect.
8. Substances which stain the gelatine, eg., neutral red and iodine, give
filtrates with lower concentrations on diminishing the pressure.
9. The rate of filtration is accelerated by amylic alcohol and butyric acid,
which accelerate the rate of sedimentation of silts in a similar way.
10. Part of the phenomena may be explicable on the mechanical view
of a blocking of the gelatine pores, but chemical relations between the
gelatine and substances filtered must be taken into consideration, and
probably the most satisfactory view is that the action of gelatine on the
solutions tested is essentially an adsorption phenomenon.
1905. | Crystalloids and Colloids through Gelatine. 331
REFERENCES.
Arrhenius and Madsen (1902), ‘ Festskrift ved Indvielsen af Statens Seruminstitut,’ IIT.
Arrhenius and Madsen (1904), ‘Bulletin de ?Académie Royale des Sciences de Dane-
mark,’ p. 271.
Bordet (1903), ‘ Annales de l'Institut Pasteur,’ vol. 17, p. 161.
Brodie (1897), ‘Journ. of Pathology,’ p. 460.
Brodie (1900), ‘ Brit. Med. Journ.,’ p. 300.
Craw (1904), ‘ Lancet,’ p. 434.
Craw (I, 1905), ‘ Journ. of Hygiene,’ vol. 5, p. 115.
Craw (IV, 1905), ‘Roy. Soc. Proc.,’ vol. 76 B, p. 179.
Ehrlich (1898), ‘ Deutsche Med. Wochenschrift,’ vol. 24, p. 597.
Ehrlich (1903), ‘ Berliner Klin. Wochenschrift,’ vol. 40, pp. 793, 825, 848.
Gatin-Grazewska (1904), ‘ Pfliiger’s Archiv,’ vol. 103, p. 281.
Hardy (1899), ‘ Journ. of Physiology,’ vol. 24, p. 158.
Krafft (1902), ‘ Zeitschr. f. physiolog. Chemie,’ vol. 35, pp. 364, 376.
Landsteiner (1903), ‘Miinchener Med. Wochenschrift,’ vol. 50, p. 764.
Martin, C. J. (1896), ‘Journ. of Physiology,’ vol. 20, p. 364.
Martin and Cherry (1898), ‘ Roy. Soc. Proc.,’ vol. 63, p. 420.
Nernst (1904), ‘ Zeitschrift fiir Hlektrochemie,’ vol. 10, p. 377.
Reid, E. W. (1901), ‘ Journ. of Physiology,’ vol. 27, p. 161.
Reid, E. W. (1904), ‘Journ. of Physiology,’ vol. 31, p. 438.
Starling (1889), ‘ Journ. of Physiology,’ vol. 24, p. 317.
Traube, M. (1866), ‘ Centralbl. f. d. Med. Wissenschaften,’ vol. 4, pp. 91, 113.
Traube, M. (1867), ‘Archiv f. Anat. u. Physiologie,’ p. 87.
332
A Case of Regeneration in Polychete Worms.
By Arnotp T. Watson, F.LS.
(Communicated by C. S. Sherrington, F.R.S. Received October 23, 1905,—Read
January 18, 1906.)
The facts recorded in the following note were ascertained in the course of my
observations made upon a marine rock-boring polychete worm found at Tenby
in the spring of the present year. This worm, a species of Potamilla, is living
in limestone rock, in which another species of the same genus, Potamilla
reniformis, is, also burrowing. It differs from the latter in various particulars,
amongst which may be noted the absence of eyes on the branchial filaments,
the colour of the blood (which is red instead of being sometimes green), the
form of the sete, and the character of the external tube, which is largely
covered, especially at the tip, with minute pieces of shell attached edgewise,
imparting to it a white, rugged appearance, somewhat similar te that of the
tube of Owenia. The worm is sometimes as much as 33 inches long, and is
seldom extracted entire from the rock, fragments only, of varying length,
usually being obtained.
It occurred to me that this material might be utilised for the study of the
regeneration of the lost parts,and my experiments in that direction succeeded
beyond my expectations. Not only did the fragments renew these parts (both
anterior and posterior) but they demonstrated the existence of a power to
economise labour in this respect, by changing the arrangement of certain of
the old parts, so as to complete the model of the original animal.
The body of the worm in question consists of a large number of segments,
all of which, with the exception of those at the two extremities, are endowed
with a set-of hooks (uncini) and bristles (sete) on either side, and it is one of
the characteristics of the Sabellide, the family to which this worm belongs,
that the character and arrangement of these appendages in the anterior or
thoracic portion differs from that in the posterior or abdominal part. The
sete in the former are situated dorsally and the uncini ventrally, whilst in
the abdominal portion the uncini are dorsal and the sete ventral. This
arrangement, besides enabling the worm to rotate on its long axis in either
direction at will, also facilitates the bringing of its thoracic ventral glandular
plates and collar-lobes into contact with the inner surface and top of the
tube, and is probably connected with the tube-forming habits of the worm.
Some of the fragments which I have had under observation were without
head and thorax, and consisted of abdominal segments only, the sete and
uncini consequently being ventral and dorsal respectively, from end to end.
A Case of Regeneration in Polychete Worms. 333
In cases of regeneration my experience has been that new segments are
much more freely. produced at the posterior than at the anterior extremity of
the body, and the problem which occurred to me was, how, and in what length
of time, the thoracic segments (about eight in number), with the inverted
arrangement of cheetal appendages, which is needful to the life-work of the
worm, would be reproduced. The answer came in the nature of a surprise.
Beside the cephalic plume-bearing segment, one new setigerous thoracic
segment only was formed, but the chetal plan of the succeeding five or nine
abdominal segments was changed; the dorsal uncini in these segments first
disappearing gave place to sete, and later the ventral setae were replaced by
uncini; the new sets and uncini, moreover, were changed to the forms
characteristic of this part of the body. In other words, so far as the chetal
plan is concerned, a new thorax had been constructed from the abdominal
segments. How far the internal structure has been affected by the change
remains to be ascertained.
The observations extended over a period of five weeks, and were made
upon two portions of apparently one and the same worm. One portion
(comprising the two parts marked @ and ¢ on the figure), about ? inch long
and consisting of 69 abdominal segments, being minus head and thorax, as
well as the anal, and numerous preanal segments; the other part, 0, } inch
long (probably the hinder portion of c), consisting of 36 preanal and the anal
segment.
In order to expose a large surface of water to the air and bring the animals
as near thereto as possible, the experiments were carried on in watch-glasses,
fresh sea-water being supplied twice daily. The first fragment, ac, in course of a
day or two, attached itself to the watch-glass by means of a narrow cord or loop
which it secreted, and which served, by a constant twisting movement of the
animal, to sever the portion, c, } inch long, and consisting of 18 of the posterior
segments. The number of parts available for observation was thus increased
to three. Each was placed in a separate watch-glass, to which it attached
itself slightly by a secretion from the ventral surface, and each part succeeded
in changing the chetal arrangement of certain abdominal segments into that
of thoracic ones, as follows—in a, nine segments became thoracic ; in ©, five ;
and in 0, nine. The normal number of thoracic segments in the few adult
specimens which have passed through my hands is eight (in one case it is
nine), but the number appears to vary considerably in any yviven species of
Sabellid.
The number of abdominal segments transformed is possibly, to some extent,
regulated by the total number of segments contained in the fragment under-
going repair, but it may also be dependent upon the number of thoracic
334 Mr. A. T. Watson. [Oct. 23,
mS
SALA A
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es) ). The cells exhibit an increase in cytoplasm, a comparative
absence of secretory activity, and a peculiar and well-defined change in the
appearance of the nucleolus. The alteration in this last-named structure
consists in its larger size and denser appearance. Furthermore, very many
nuclei are to be seen in a state of active division. Whilst some exhibit
various stages of mitosis, others are clearly undergoing fragmentation or
amitosis.
At this stage of the development of the tumour, the peripheral cells that
are dividing mitotically show all the characters of ordinary premaiotic
divisions, and the normal number (32) of chromosomes can frequently be
counted with certainty (figs. 5 and 10). But concomitantly with the first
changes indicated in the epithelial cells at the edge of the neoplasm, a marked
activity may be observed to take place on the part of the leucocytes. These
bodies are seen to be in a condition of active migration and multiplication,
much like that which occurs during the early stages of simple inflammation.
In the subsequent stages, however, the early parallelism with inflammatory
processes is lost, and there supervenes a remarkable phase in the further
development of the cancerous cells. Not only do the cells of the tissue in
question multiply with great rapidity, whilst the leucocytes amongst them
are enormously increased in number, but the latter are seen not infrequently
to force their way into the cancer cells, particularly in the so-called “giant
cells,’ where, however, they are still to be recognised with ease and certainty
(figs. 1,2, d,11,12,13). This circumstance has already been noticed by others,
but we have been led to attach a somewhat special importance to its occurrence.
Some writers have suggested that the cancer cells are acting phagocytically
upon the leucocytes, but, as a matter of fact, the further sequence of events
indicates that the cancer cells are no more to be regarded as attacking the
leucocytes than the latter as destroying the cancer cells. There can be no
possible doubt that the leucocytes actively force their way into the elements
in question. They may not seldom be observed to be in close juxtaposition
with these, or in a hollowed depression, or finally they may be discovered just
within the cell membrane, where they are easily recognised on account of
their characteristic nuclei (fig. 11). They show no sign of disintegration—at
least, in the great majority of cases—and the fact that they may persist for a
considerable time without destroying the cell into which they have invaded,
is proved by examples in which a leucocyte lying in the cancer cell is seen to
be surrounded by several nuclei that have clearly originated by the frag-
mentation of the original cell nucleus, and, indeed, one of these is shown to
be still dividing amitotically.
1905. | On the Cytology of Malignant Growths. 341
But the strongest proof of the persistence of the leucocyte under these
remarkable conditions is afforded by the cases, not few in number, in which we
have been able to trace the leucocyte actually dividing within the cancer cell
(figs. 12,13). Of course, it is only during the early stages that it is possible to
be certain that a second dividing nucleus in a mass of protoplasm belongs to a
leucocyte, and does not represent mitosis in a small nucleus that has arisen
by fragmentation. But we have seen so many cases of early stages of leuco-
eytic mitosis within the cancerous (or “precancerous”) cell that it seems
impossible to resist the inference that many of those frequently occurring
cases in which a small nucleus is seen in the later phases of mitosis within the
large nucleated cancer cell are to be attributed to this source. The nuclei of
the cancer cell and leucocyte often divide simultaneously, and the two nuclear
figures may also coalesce more or less intimately, and thus a commingling of
leucocytic and epithelial chromosomes occurs on a spindle that becomes
common to the two nuclei concerned. The cells so affected were, as already
stated, usually the very large (giant) cells so characteristic at this stage of the
development of the tumour, and we found that more than one leucocyte might
enter and persist in a single cancer cell. In the earlier stages, of course,
there is no difficulty in clearly recognising the intruding cell, since it retains
its own cytoplasm and lmiting membrane intact (see fig. 11), and the highly
characteristic structure of the nucleus enables it to be identified even after
these criteria have ceased to exist.
In the same region in which this series of events is proceeding a number of
cancer cells are to be seen in various phases of mitosis, and, both in the aster
and diaster of such nuclei, larger numbers of chromosomes were often
encountered than are proper to normal somatic cells. These increased
numbers are partly to be ascribed to the pluripolar mitosis distinguished by
Hertwig and by Von Hansemann, and they result from the simultaneous
mitosis of a number of nuclei lying in a common cytoplasmic mass.
But the observations recorded above indicate that, in the addition of
leucocytic nuclei to those of the actual epithelial cells, we have confronted,
at any rate, with one of the sources to which these excessive numbers of
chromosomes (hyperchromatic nuclei of Von Hansemann) may be attributed,
although a large number of the cells continue to multiply in the manner
already described, it may also be seen that there exists a very considerable
amount of amitosis, or direct nuclear divisions in the cells of the young parts
of the tumour. There appears to be no evidence which would point to the
conclusion that amitosis is in any way bound up with degeneration, or
diminishing activity in those cells in which it occurs. Elements that have
previously multiplied by amitosis and by fragmentation have given rise to
342 Prof, Farmer, Messrs. Moore and Walker. [Nov. 17,
the highly characteristic multinucleate cells, may again assume the mitotic
method of increase, and vice versd. A curious feature in the further division
of these multinucleate cells, or syncytia as they may, perhaps, be more
appropriately termed, is seen in the almost invariable circumstance that,
on the resumption of mitotic activity, all the nuclei are in exactly the same
phase.
This simultaneous character of the process is one which is shared by
many other syncytia, eg., the myxomycetes. In these organisms, the nuclei
are commonly observed not only to be dividing simultaneously over a
considerable area of the plasmodium, but they also exhibit identical phases
of the process at any given time. In examples of this simultaneous mitosis
within the neoplastic syncytia, it often happens that the spindles of some, or
even all of the dividing nuclei, become more or less intimately fused
together, and in this way various forms of pluripolar mitosis are produced.
Probably these pluripolar divisions owe their origin chiefly to the cause just
indicated.
The figures produced are extremely variable, and it not unfrequently
happens that, whilst the chromosomes belonging to the different nuclei
are ageregated in the centre, the poles of three or more of the spindles
involved are quite separate. In other examples the groups of chromosomes
do not coalesce, but each equatorial plate is quite distinct, and lies in a plane
different from that occupied by the equatorial plates of the other spindles.
But when a more intimate fusion of the ends of two or more spindles takes
place, it is obvious that the daughter nucleus formed in relation to such
unions will receive an excessive number of chromosomes.
We would call special attention to the fact that giant cells of this
character, also containing several nuclei, are present not only in the normal
human testis, but also in the so-called red bone marrow, and that pluripolar
mitosis may occur in such cells in a manner precisely similar to that so
characteristic of cancerous tissue. The divisions of these early cancerous
cells also exhibit other characters likewise encountered in the cells of the
testis. Very often the daughter chromosomes do not move regularly towards
the poles, but some either stray out of the direct line, or in other ways
occupy unusual positions. These figures are also well known to occur in the
heterotype division of some spore mother-cells of plants. In yet other
examples of divisions in cancerous tissues, we have confirmed the observation
of Von Hansemann that some of the chromosomes, as they are passing to the
spindle poles, get ahead of their fellows, and form isolated or grouped
chromatic particles that look as if they are about to be left out in the
cytoplasm when the daughter nuclei become reconstituted. These figures
1905. | On the Cytology of Malignant Growths. 343
are also paralleled by similar occurrences that may be seen in the cells of the
testis,and they are known to occur during the maiotic divisions of some plants,
It is thus evident that hyperchromatic nuclei, that is, nuclei containing an
excessive number of chromosomes, may be produced in at least two ways:
firstly, by the inclusion of leucocytes, and the incorporation of the chromo-
somes belonging to these bodies with those of the cancer cells when mitosis
sets in; secondly, through the formation, whether by amitosis or mitosis, of
multinucleate syncytia, and by the subsequent confusion and mixing of the
chromosomes originally belonging to two or more of the nuclei when the
equatorial plate stage is reached.
These aberrant modes of division are found to proceed concurrently with
the normal somatic mitoses that are going on in other cells in their immediate
vicinity. It is impossible to say definitely whether there may exist any sort
of alternation between the two types, though we are inclined to think that
such is not the case. It is, however, important to notice that all the mitoses
described above, whether they are normal in the number of chromosomes
or not, agree in conforming to the somatic type of division. That is to say,
no matter how many or how few the number of chromosomes involved may
be, the spireme eventually divides into a number of rod-like elements, each
of which splits longitudinally, and the daughter chromosomes resulting from
such fusion are severally distributed between the daughter nuclei finally
produced. In such typical cases this of course means that each of the two
daughter nuclei receives one longitudinal moiety of such original chromosome.
But as we pass inwards from the growing edge of the tumour we encounter
cells in which the nuclei exhibit important deviations from the ordinary
somatic type of mitosis, and exhibit the characters otherwise met with during
the heterotype division (¢/. figs. 6, 7, 8). In the early stage of the phase of
such nuclei the spireme exhibits that characteristic bunched appearance
recalling the well-known contraction figure that is normally to be seen at the
onset of the maiotic phase, that is in the prophase of the heterotype mitosis,
in animals and plants. In addition to this, we have been able to ascertain
that at about the same stage the spireme thread exhibits the longitudinal
fission (fig. 6) that is highly characteristic, though perhaps not exclusively
confined to the prophase of the heterotype division. The fission is especially
well seen in those cases in which a marked polarisation of the spireme is
apparent. But the most striking evidence of the validity of the comparison
that we drew in 1903 between these particular nuclei and those of the
reproductive cells during the maiotic phase of the animals and plants does not
depend solely on the similar mode of evolution of the chromosomes from the
resting nuclei in the “gametoid” cancerous and the true reproductive
VOL, LXXVII.—B, 2¢
344 Prof. Farmer, Messrs. Moore and Walker. [Nov. 17,
elements. The number of the chromosomes furnishes a far more important
criterion. It is seen that a large number of dividing nuclei contains less than
the normal complement of chromosomes. We have made a number of careful
counts of the chromosomes in numerous cases of carcinoma, and always with
the same result. In especial, we are indebted to Mr. L. Robinson for his
assistance in this somewhat trying task. He has estimated the chromosomes
in 400 dividing nuclei, taken (100 from each) near the actively growing
regions of three different carcinomata originating respectively from the rectum,
scrotum, penis, and in an example of deciduoma malignum.
In every case we find two well-defined maxima, one set of nuclei containing
32, the other 16, chromosomes. For purposes of comparison he has counted
chromosomes of the testis of the cockroach (Curve F), so as to obtain a control
indicating the probable degree of accuracy represented by the estimations in the
cancer nuclei. The same two maxima are, of course, apparent, but there is a
similar average error around the maxima, due to the difficulty of the actual
counting, and also the chance that some of the chromosomes might be absent
from the section, or that a limited degree of variation may really occur. And,
having regard to the fact that in the human species the chromosomes are not
easy, even under favourable conditions, to estimate very accurately, whereas
in the case of the cockroach the observer encounters far less difficulty in
this respect, the results may, we think, be described as satisfactory. For
although, after what we have said, it is obvious that, owing to amitosis,
and especially to pluripolar mitosis, a considerable extent of variations is
to be anticipated, the grouping of the numbers around the maxima of
32 (somatic) and 16 (reduced) is quite unmistakeable, as 1s shown in the
accompanying curves.
ie
ee |
Number of cells
Carcinoma of Rectum.
The ordinates represent the number of cells that contained any given number of
chromosomes, as indicated by the abscissz.
A. Cancer of the Rectum.—The hypochromatic nuclei to the left somewhat obscure, the
maximum at 16. The grouping of numbers about 24 and 64 are fairly well
shown.
1905. | On the Cytology of Malignant Growths. 345
in
lad (i, anni
Number of ch eae
2 |
Number of cells
Epithelioma of the Scrotum.
Number of cells
CI
10 15 20 25 30 35 40 45 50 55 60
Number of chromosomes
Epithelioma of the Penis.
a te”
AlN tit | IS
Io I5 i 40
Number oF Eirencseen
5
un
Number of cells
°
—
Deciduoma malignum.
B. Epithelioma of the Scrotum. The maxima about 16, 24, and 32 are distinct, that
about 64 not so clear.
C. Epithelioma of the Penis.—The maxima in the regions of 16, 24, 32, 48, and 64 are all
fairly distinct.
D. Deciduoma malignum.—There is considerable irregularity in the nuclei in this growth,
which was somewhat advanced, and deviations are therefore to be anticipated.
Clee
Namommeielyeemasciwl emp b |
Mec ee fede |
Nest
ANA HN | | LAM
AANA ee DectoohluNh
wNueer oF dn Bieosance
E. Combined Curve drawn from the Results shown in the preceding Four Cases of Cancer,
viz., carcinoma of rectum, epithelioma of scrotum and penis, and deciduoma
malignum, The three maxima about 16, 32, and 64 are unmistakeable.
DG D
4
25
20)
15
am
fe)
eh
Number of cells.
346 Prof. Farmer, Messrs. Moore and Walker. [Niowaraltyis
30
25
pce}
Number of cells
w
8e
is 20 25 30 35 40
umber of chromosomes z
z
Testis of Periplaneta Americana.
F, Curve obtained by countings made from dividing nuclei of the maiotic and premaiotic
cells of the testis, in order to estimate the probable error in the far more difficult
cases of cancer. It will be seen that there is some not inconsiderable variation
about the two maxima of 16 and 32. This is due partly to underestimating the
number of chromosomes actually present, and partly to the nuclei having in some
cases been partly damaged in preparing the section.
We shall further consider this matter in the concluding part of the paper:
at present we are mainly concerned with showing that there exists a
striking resemblance between what we have termed the “gametoid” cells of
cancer and the cells of normal reproductive tissues, and as we pass to the
later phases of mitosis we find the same loop and barrel-shaped chromosomes
present in both, and we have occasionally seen, during the diaster of a cancer
nucleus, the late longitudinal fission in the daughter chromosomes as they
diverge from each other, just as it occurs in the heterotype diaster of so
many animals and plants. An inspection of the curves shows the relative
frequency of the different numbers of chromosomes met with in the
younger cancerous areas. Whilst, as already pointed out, the two maxima of
16 and 32 are unmistakeable, it is also obvious that amongst the generally
irregular numbers two other groups occur with greater frequency than others.
Thus, there is a distinct indication that nuclei containing about 24 chromo-
somes may be regarded as forming a distinct group, also that a second, though
far less well-marked, series is characterised by containing about 64 (double
the normal somatic number) chromosomes. It may be that the latter are
related to the ingression of the leucocyte already described, but it is difficult
at present to guess at the significance of the grouping of 24. There is no
obvious indication that the nuclei with 48 chromosomes are specially common,
and, in the absence of more direct evidence, it is useless to indulge in
speculations that may prove to be devoid of all foundations.
1905. | On the Cytology of Malignant Growths. 347
In animals, as has already been stated, it invariably happens that, after the
onset of the first maiotic (heterotype) mitosis, there ensues only one further
nuclear division, commonly designated as the homotype, on account of its
close general resemblance with a normal somatic mitosis. The principal
point of constant difference lies in the retention in the former of the reduced
number of chromosomes. The cells originating from this division give rise
after a more or less complex series of changes of form and of the inter-relation
of their constituent parts and the sexual cells without any further intervening
nuclear divisions. In plants this is not the case. The cells issuing from the
homotype mitosis always undergo one or (often) many subsequent divisions
beforé some or all of the resulting units develop into sexual cells. It is
therefore of interest to find in cancerous tissue that there is abundant
evidence that the cells, the nuclei of which have undergone reduction, are
capable of continued division, and, indeed, a great part of the tissue of the
cancer is made up of such cells, which, in accordance with the terminology we
have elsewhere employed, we may term post-maiotic, or “gametoid.”
It will be seen that we differ from Von Hansemann in our explanation of
these “hypochromatic” nuclei, regarding them as have arisen, not as the
author just named believes, by a dropping-out of chromosomes from the
spindle, or through some form of degeneration, but chiefly as the result
of a process resembling, or identical with, that by which reduction is
ordinarily effected in the tissues destined to give rise to the gametic cells.
But we desire to definitely state that, in using the term “gametoid,” we
expressly differentiate between the cancerous cells and those of normal
reproductive tissues. The relation existing between them, if any, is at
present obscure ; and, though we think the resemblances, which will be still
further emphasised by facts we are about to describe, are very suggestive, we
are far from holding the views which have been expressly or implicitly
ascribed to us by other writers as to the identity of gametic with “ gametoid”
cells and tissues.
Finally, then, it is clear that there exist in the facts of pluripolar mitosis,
on the one hand, and in amitosis on the other, a mechanism sufficient to
explain all the irregular numbers encountered in a young cancer. But the
irregularities, while masking, cannot conceal the far more frequently recurring
numbers of chromosomes, whereby the reduced (halved) and, though far less
frequently, the double, numbers become apparent. But the existence of the
irregularities indicated above often renders extremely difficult the task of
deciding to what category a particular departure from the normal somatic
number is to be relegated.
There is a further body of evidence bearing on the resemblance between’
348 Prof. Farmer, Messrs. Moore and Walker. [Nov. 17,
cancerous and normal reproductive tissue to be derived from a study of the
so-called Plimmer’s bodies of cancer.*
It was shown by one of us in 1895f that, during the prophase of the first
maiotic (heterotype) division of the spermatogenetic cells of mammals, the
archoplasm undergoes a peculiar and definite series of metamorphoses. In
ordinary somatic or premaiotic cells, this body is seen to le beside the nucleus
as a dusky mass of protoplasm, in the centre of which are found the centro-
somes. Thus, in these cells, the attraction sphere consists of the archoplasm
plus the centrosomes (fig. 3, 0, fig. 4, a).
When, however, we turn from the premaiotic or the somatic cells to
the prophase of the heterotype (first maiotic) mitosis, we find these’ two
constituents have become separated (fig. 4, b). The centrosomes migrate from
the centre of the archoplasm, and are eventually seen to le outside that body,
and*completely detached from it (fig. 4, ¢). At the same time the archoplasm
itself undergoes a change, small vesicles are developed in its substance (fig. 14),
and, at the close of this particular cell-generation, both vesicles and archo-
plasm become merged and lost in the general cytoplasm of the daughter cells.
In the prophase of the second maiotic (homotype) mitosis the same peculiar
phenomena recur, and the archoplasm and the vesicles, in lke manner,
become lost during the later stages of this (homotype) division. In the
spermatids, which result from it, the persistent centrosomes can be readily
seen to be perfectly disconnected with the new archoplasm which is
differentiated in these cells. The archoplasm becomes filled with minute
vesicles, as in the two preceding cases, subsequently the vesicles enlarge,
and they either fuse together, as in some mammals, or one usually takes the
lead and grows larger than the rest, as commonly happens in the guinea-pig
and in man (fig. 15). The body thus formed was originally termed the
archoplasmic vesicle in 1895,t and it is a very conspicuous and constant
feature peculiar to the sperm cells of the vertebrata, whilst it has also been
encountered by various observers in animals outside that group.
When fully developed, the archoplasmic vesicle often assumes a size
approximating to that of the nucleus itself, the latter being often deformed
into a crescentic shape, owing to the enlargement of the vesicle that hes
adjacent to it in the cell. In normal spermatids, the vesicle and its contents
ultimately form the so-called “cephalic cap” of the spermatozoon (fig. 16, @).
* See ‘Roy. Soc. Proc., vol. 76 B, “On the Resemblances existing between the
‘Plimmer’s Bodies’ of Malignant Growths and certain Normal Constituents of Repro-
ductive Cells of Animals,” by J. Bretland Farmer, J. E. 8. Moore, and C. E. Walker.
+ Moore, ‘ Internat. Monatschr. f. Anat. u. Physiol.,’ 1894.
{t Moore, loc. cit.
1905. | On the Cytology of Malignant Growths. 349
Now, the “Plimmer’s bodies” are well known in the cells of many
cancerous growths (fig. 17), and they are most commonly met with in the
young erowing portions of the tumour. They appear in the form of vesicles,
and consist eventually of a fairly well-defined wall, enclosing a clear space, in
which is suspended a smal] and densely refracting granule. They appear to
occur with greater frequency in cancers of a glandular or glandular-epithelial
origin.*
They lie in the cytoplasm of the cancer cells, usually in close proximity to
the nucleus. They vary im size from excessivery minute bodies to forms as
large as the nucleus itself. The special interest attaching to the Plimmer’s
bodies depends on the fact that they have commonly been regarded as
peculiar to cancer cells, although Hondat believes that he has occasionally
encountered them in inflammatory tissue. They have, in fact, been variously
interpreted. Some investigators have regarded them as parasitic organisms,
more or less intimately connected with the etiology of the disease, whilst
others have seen in them a differentiation of the cancerous cell itself.
Borrelf suggested that they might represent hypertrophied centrosomes, but
the observations of Benda,§ who showed that centrosomes and Plimmer’s
bodies coexisted in the same cell, have rightly been held to disprove the view
advanced by Borrel.
When the foregoing facts are all taken into consideration, the case
originally upheld by ourselves|| appears to be a strong one. We see no
escape from the position that the Plimmer’s bodies of cancer represent the
archoplasmic vesicles that occur in the normal reproductive cells at the
stages already indicated. And this forms an important link in the chain
of similarities connecting cancerous tissue with the normal reproductive
elements. But in this relation it is of interest to note that we have recently
observed bodies, which appear to be closely similar to archoplasmic vesicles,
to occur at apparently definite stages in the life history of certain leucocytes
which are present in bone marrow.
General Conclusions.
To sum up the observations already recorded in this paper, it may be
seen :—
* Greenough, ‘Third Report of the Caroline Brewer Croft Cancer Com.,’ Hary. Med.
School, 1905.
+ Honda, ‘ Virch. Arch.,’ vol. 174.
t Borrel, ‘Ann. Inst. Past.,’ vol. 15.
§ Benda, ‘ Verh. deutsch. Gesellsch. f. Chir.,’ 1902.
|| ‘Roy. Soc. Proc.,’ vol. 76 B, pp. 230 et seq.
350 Prof. Farmer, Messrs. Moore and Walker. [Nov. 17,
1, That a primary growth originates in the first instance as the result of
a change in the nature of a number of previously functional somatic cells.
2. That the transformation may affect a considerable number of cells, and
certainly continues to operate for some time.
3. That, as the result of the change, mitotic and amitotic activity is
awakened, and proceeds rapidly, with a consequent increase in the mass of
affected tissue.
4. That during this increase a remarkable activity prevails amongst the -
leucocytes, at first resembling that seen in inflammatory processes, but finally
leading to the union of at any rate some of the affected cells with one or more
leucocytes.
5. That in the subsequent divisions of these cells the nucleus of the
leucocyte divides simultaneously with that of the cancer cell, and their
chromosomes may become mingled in cleavage figure.
6. That multinucleate cells (syncytia) may arise by mitosis or by amitosis,
unaccompanied with the division of the mass of protoplasm.
7. That the resulting nuclei may divide normally and mitotically, or the
nuclear figures may be more or less mingled, and hence all sorts of variations
in the number of chromosomes may occur. But the mode of chromosome
evolution and division follows the somatic type.
8. In addition, a form of mitosis occurs, leading to nuclei with half the
number of somatic chromosomes, and the phases closely accord with those
observed during the heterotype (first maiotic) mitosis of animals and plants.
9. Subsequent divisions occur, in which the reduced number of chromo-
somes is retained, the type of division otherwise resembling that of ordinary
somatic cells. These mitoses fall into the category corresponding with the
post-maiotic mitoses of plants.
10. During the maiotic and post-maiotic divisions in the cancerous cells,
structures are present which have been designated as Plimmer’s bodies.
These are common to cancerous cells and to the reproductive cells of the
testis at a particular phase in their evolution. The only other cells in which
structures resembling the bodies in question have been observed are possibly
those forming certain of the leucocytes in bone-marrow.
It will be evident from the above summary that the change from the
healthy to a cancerous development is intimately bound up with definite
change in the cells affected. The onset of the change is probably to be
attributed to the operation either of new stimuli upon the body cells, or to a
change in the constitution of the latter. Such an alteration might originate
in a variety of ways. For example, it might be ascribed to the influence of
a parasite. But we have never succeeded in tracing any such cause, and it
1905. | On the Cytology of Malignant Growths. 351
becomes necessary therefore to seek for some other explanation for the
phenomena actually witnessed.
It is quite certain, in the first place, that we are dealing with the trans-
formation of functional somatic cells into cancerous ones, and this, to our own
minds, affords a complete refutation of the hypothesis as to the persistence of
“embryonic rests,” such as have been supposed by Cohnheim and his followers
to account for the incidence of the disease.
We have drawn attention to the events that occur in connection with the
invasion of the cells of the young growths by leucocytes, and, although we
are fully aware that further investigations into the details of these processes
are required before a final opinion can be expressed as to their true significance,
the facts themselves are very suggestive.
Furthermore, the interest attaching to these fusions is not lessened by a
study of the bone-marrow, in which the leucocytes can be most advantageously
observed. For we have seen in this tissue all the abnormal types of nuclear
and cellular division that are so highly characteristic of cancerous cells, and
we have ascertained a fact of even greater importance, namely, that some
of the nuclei of dividing marrow cells certainly possess less than the full
complement (52) of somatic chromosomes. We would, further, lay emphasis
on the occurrence, in the same preparations of bone-marrow, of other cells in
which the process of mitosis was strictly somatic in character, both as regards
the form and number of the chromosomes. But it is none the less certain
that the other nuclei exhibit chromosomes of a remarkable form, elongated in
the direction of the spindle, and strongly resembling those which are so
characteristic of the heterotype mitoses of the testis or of a cancer.
Whilst it is obvious that further investigation on the cytology of bone-marrow
is urgently needed, it is evident that, if it should ultimately prove that the cells
which are derived from the results of fusion of a leucocyte with a tumour
cell really represent the progenitors of the malignant elements themselves, a
satisfactory explanation would be afforded not only of the striking nuclear
character of the diseased tissues, but also of the invasive and destructive
powers they undoubtedly possess. The destructive action of the leucocytes
themselves on other cells of the body, especially during old age, is too well
known, owing especially to the valuable researches of Metschnikoff, to call
for further comment here.
Such a view of the case as is here tentatively suggested is not in
conflict with the idea embodied in the term “gametoid” tissue, but rather
forms an extension of it. We have, as already pointed out, from the first
maintained the existence of a resemblance, extending to extraordinarily
minute detail, between the “ gametoid,” cancerous, and the reproductive tissue,
352 Prof. Farmer, Messrs. Moore and Walker. [Nov. 17,
which, in the case of animals, gives rise to the gametes immediately after
maiosis. But it is also now certain that there exist certain striking
similarities between the leucocytic and reproductive cells which are, in
themselves, highly suggestive, and this is not diminished by a consideration
of the earlier phylogenetic history of wandering and reproductive cells in
more primitive animals, for example, in sponges.
For the present, however, and in the absence of more complete and accurate
knowledge on the evolution of the leucocytes, we may close by remarking
that the various peculiar characteristics of cancerous cells find thei closest
analogies in the cytological processes that are exhibited in the formation of
the reproductive cells, and in those maiotic phenomena that so especially
distinguish them.
DESCRIPTION OF PLATES.
PLATE 8.
Fig. 1.—Section of the growing edge of a young Carcinoma of the Rectum.
lw, 1y,1z. Enlarged parts of the same drawing. The letters c, d,e, correspond with
those on the main figure.
a. The portion to the right represents the normal structure of the rectum ; 6 the zone
in which transmutation from healthy to cancerous tissue is proceeding.
c. Cell showing somatic division (see also 1 z).
d. Cells in this zone containing leucocytes (see also 1 z and 17).
e. Cell showing prophase of first maiotic (heterotype) mitosis (see also 1 2).
f. Cut portion of crypts, but belong to the zone of transformation.
g. Portions of the growth invading the adjacent layers.
PLATE 9.
Fig. 2.—Section through young Epithelioma of the Penis.
2x, 2y, 22. Enlarged parts of the same drawing. The letters a, b, c, d, correspond with
those of the main figure.
a. Cells showing somatic (premaiotic) divisions.
b. Cells showing somatic division, but with excessive number of chromosomes.
c. Cell showing first maiotic (heterotype) division.
d. Cell with leucocyte in its cytoplasm.
-
Puate 16.
Fig. 3.—Small Portion of the Testis of a Guinea-pig, showing (a) premaiotic cell dividing ;
(b) ceil in prophase of first maiotic (heterotype) division. In this it will be seen that
the centrosomes are at the centre of the archoplasm.
Fig. 4.—Portion of the Testis of a Guinea-pig, showing (a) cells with the synaptic contrac-
tion, and the normal condition of the attraction sphere ; (6) late stage in the prophase
of the first maiotic division, showing the centrosomes detached from the archoplasm ;
(c) homotype prophases showing. The same dismembered condition of the attraction
spheres.
Fig. 5.—Cell from the early Cancer of the Rectum given in fig. 1, showing the somatic
character of division. Compare with fig. 3, a.
Fig. 6.—Cell from Cancer of Rectum given in fig. 1, showing the characters of the
prophase of the heterotype division. Compare with fig. 4, a.
*
Farmer, Moore and Walker.
Roy. Soc. Proc., B. vol. 77, Plate
ues il.
i he
a 2ae
Ny Feadeguol (Biber
Ove Oy © 595°
- y 7 ue Lf) YO
W541 Ba ee,
lite, 2.
Farmer, Moore and Walker. voy. Soc. Proc., B. vol. 77, Plate 10.
Na fates:
Farmer, Moore and Watker. Roy. Soc. Proc., B. vol. 77, Plate 11.
Soc. Proc., B. vol. 77, Plate 12.
Roy.
Farmer, Moore and Walker.
Fig. 4.
1905. | On the Cytology of Malignant Growths. 353
Fig.
PuatTe 11.
7.—Cell from an example of Decidua malignum, showing the later phases of the
heterotype mitosis.
. 8.—Similar Cell from an Epithelioma of the Tongue.
. 9.—Cell from the Testis of Man, showing the later stages of the heterotype division.
Compare with figs. 7 and 8.
. 10.—Cell from a Cancer of the Rectum, showing the somatic or premaiotic character
of the chromosomes and the large number of these elements.
. 11.—Cell from an early Cancer of Rectum, showing the peculiar condition of the
nucleus, which suggests amitosis ; also two leucocytes (a) within the cytoplasm.
. 12.—Cell from the same showing nucleus in the prophase of division, and also an
intruded leucocyte (@), with its nucleus in the same phase.
. 13.—Cell from Cancer of the Rectum, showing nucleus in division, and that of
intruded leucocyte (@) in a late prophase.
PLatE 32.
. 14.—Portion of the Testis of a Guinea-pig, showing spermatids with developing
archoplasmic vesicles and centrosomes.
. 15.—Portion of the Testis of a Guinea-pig, showing a later stage in the development
of the archoplasmic vesicle. In this the origin of the tail of the spermatozoon is also
seen, in connection with one of the centrosomes.
. 16.—Portion of the Testis of a Guinea-pig, showing the remains of the archoplasmic
vesicle becoming converted into the so-called “cephalic cap” (a) of the spermatozoon.
. 17.—Cells from a Cancer of the Breast, showing Plimmer’s bodies and the position of
the centrosomes. Compare with figs. 13 and 14.
304
On the Sexuality and Development of the Ascocarp of
Humaria granulata Quél.
By Vernon H. Buiackman, M.A., Assistant, Department of Botany, British
Museum; Late Fellow of St. John’s College, Cambridge ; and HELEN
C. I. Fraser, B.Sc., Assistant Lecturer, Royal Holloway College.
(Communicated by Professor Marshall Ward, F.R.S. Received October 31,—Read
December 14, 1905.)
[Puates 138—15.]
The observations of Harper(15, 16, 17) on Spherotheca, Erysiphe and
Pyronema, have clearly shown that some at least of the Ascomycetes exhibit
an ordinary sexual process. It is true that attempts have been made by
Dangeard (10) to refute Harper’s observations, and doubt has been cast on
his work by Lindau (22), Holtermann (18), and others; but the recent very
convincing work of Claussen (8A) on Boudiera,* together with the strong
circumstantial evidence obtained by Barker(1 and 2} in Monascus and
Ryparobius, by Miss Dale(9) in Gymnoascus, and by Baur(3, 4), and
Darbishire (104) in lichens, and, also, the confirmation by ourselves (7) of
Harper’s work on Spherotheca, leave no doubt that the sexuality of the
Ascomycetes is founded on a firm basis.
The earlier non-cytological observations of a number of forms, however, .
have shown clearly that the existence of a normal sexual process can hardly
be expected in all the Ascomycetes. For example, in Melanospora parasitica
Kihlmann (20) observed the development of the archicarp into the
perithecium without the intervention of an antheridium; in Chetomium,
Oltmanns (24) found that the antheridium was usually absent; in Ascobolus,
the earlier observations of Woronin (25) and Janczewski (10), and the later
observations of Harper(16), brought to light no definite antheridium.
Again, among the lichens, according to the observations of Fiinfstuck (13),
in Peltigera and Peltidea the ascogonia are without trichogynes ; and Solorina
saccata, according to Baur’s (4) researches, seems clearly to develop without
any ordinary fertilisation. It is obviously, then, very desirable that the
cytology of some member of the Ascomycetes, the ascocarp of which
develops without fertilisation by an antheridium, should be carefully
investigated. The form here studied is of this type and hence is of peculiar
interest.
Humaria granulata Quél (= Peziza granulata Bull), a common Dis-
* The form investigated by Claussen would seem to be more correctly placed in the
genus Ascodesmis (vide Fr. Cavara, ‘ Annales Mycologici,’ vol. 3, 1905, p. 363).
Sexuality, etc., of the Ascocarp of Humaria granulata. 355
comycete about 5 mm. in diameter, and of a yellow, orange or reddish tint,
is found growing on the dung of various animals, especially of cow, and
is most abundant during autumn and winter. The spores, apparently,
normally germinate only after they have passed through the alimentary
canal of the animal, for artificial cultures could not be obtained. A
preliminary peptic or tryptic digestion or a combination of both seemed to
have no effect on germination. Only a small number of experiments were
made in this direction, for by bringing the material into the laboratory,
natural cultures can sometimes be obtained, in which the fungus occurs in
such abundance in appropriate stages that the necessity for artificial cultures
is completely obviated.
The material was chiefly fixed in Fleming’s weak fluid, which was allowed —
to act either for 24 hours or for one hour, fixation being completed in the
latter case with Merkel’s fluid. Either safranin, gentian violet and orange,
or Benda’s iron-hematoxylin were used for staining. The very youngest
stages of the apothecia are of course quite invisible, even with a powerful
hand-lens, but sections of them were secured by removing and fixing the
superficial layers of the substratum on which young apothecia were just
visible. The behaviour of the closely-packed nuclei of the ascogonium was
best followed in sections 4 w in thickness.
Vegetative myceliwm.—The vegetative mycelium consists of cells which
show numerous nuclei, but these, unlike those of the ascogonium, are not
at all well marked, but appear generally as slightly staining homogeneous or
granular bodies which sometimes show a minute nucleolar dot (Plate 13, fig. 1).
The cells of the whole vegetative mycelium and of the apothecium contain
a number of fairly large spherical granules, which stain deep red with the
safranin of the triple stain. Im all the hyphe of the vegetative mycelium
and many of those of the ascocarp, these granules are found collected in
eroups on opposite sides of the transverse walls (figs. 1 and 4). These
groups of granules were observed by Woronin(25) in this form and in
Ascobolus, and by Harper (17) in Pyronema, but their function is unknown:
Harper has suggested that they may have something to do with the passage
of material from cell to cell through the wall. No reproductive organs other
than the apothecia were observed in connection with this form.
Development of Apothecium.
As long ago as 1866, Woronin (25) showed that the apothecium began by
the development of an archicarp as a side branch of an ordinary hypha of
the mycelium. He observed that the apical cell of this branch was round
and very much swollen, and that, later, side branches grew up from the cells
356 Mr. Blackman and Miss Fraser. Sexuality and [Oct. 31,
of the stalk and completely invested the apical cell. The large cell he
considered to be of the nature of an egg, while one of the branches growing
up from below he thought was probably of the nature of an antheridium:; he
was unable, however, to follow any further details of development.
Actual Observations.—As Woronin observed, the first beginning of the
archicarp consists of a branch with a variable number of somewhat short
cells (figs. 2A, 2B, 3). The apical cell of this row is the ascogoniwm* which
soon increases in size and becomes spherical, and exhibits beautiful vacuolate
structure (figs. 24, 2B). The lower cells also increase in size and both they
and the ascogonium become closely filled with food-material, so that the
whole archicarp has a dense and opaque appearance (figs. 4, 5). Before the
ascogonium has attained its full size a number of narrow branches begin to
erow out from the cells of the stalk immediately below (fig. 4). These are
the first beginnings of the investing hyphe which soon grow up and com-
pletely cover in with layers of plectenchyma the ascogonium and upper few
cells of the stalk (figs. 5,9). In cleared preparations, however, the cells of
the ascogonium and of the stalk, owing to their greater density, can, for a
time, be distinctly seen threugh the investing sheath (fig. 5).
The number of cells in the stalk is variable ; there may be only a few, as
is apparently the case in fig. 3, but usually a large number are to be
observed, as in fiy. 5. From one of the cells about the middle of the stalk a
small side branch was sometimes seen which grew down into the substratum
and apparently aided the stalk in absorbing nourishment.
None of the hyphe which grow up from the stalk act im the way Woronin
suggested; they are all mere vegetative investing hyphx, no antheridiwm
being developed.
The ascogoniuw shows a vacuolate protoplasm with a number of nuclei
which are better defined than those of the vegetative cells; with the growth
of this organ these nuclei become much more distinct, exhibiting a nuclear
membrane and a single deeply-staining nucleolus (figs. 6, 7), but no
chromatin is to be observed in the nuclear cavity. As development proceeds
the nuclei increase only slightly in size but enormously in number ; and the
small vacuoles are replaced by one or more large ones (figs. 8, 10). At about
the stage when the vegetative hyphe completely surround the ascogonium,
the wall of the latter becomes thickened and shows a distinct differentiation
into two layers, the outer, thin and deeply staining, the inner, thicker and
lightly staining (figs. 10, 11).
' The wall between the ascogonium and the uppermost stalk cell exhibits at
a young stage the usual apposed groups of granules, but at a later stage the
* For a discussion of this use of the terms archicarp and ascogonium, vide infra.
1905.| Development of the Ascocarp of Humaria granulata, 357
granules apparently fuse together, for when the ascogonium has reached its
full size this wall shows two large and deeply-staining masses placed
opposite one another on either side of the wall (fig. 13). The masses
sometimes show a central deeply-staining portion, and an outer, irregular,
less dense portion (fig. 13). When the ascogonium and stalk cells become
emptied these masses disappear. Besides the special accumulation on the
walls a number of large granules are usually to be found scattered in the
cytoplasm of the ascogonium and stalk cells (fig. 9).
When the ascogonium has become covered in with several layers of vege-
tative hyphe the ascogenous hyphe appear as narrow, thin-walled outgrowths
from the thick-walled ascogonium, and make their way through the close
mass of investing hyphe (figs. 10, 11). Into the ascogenous hyphe there
pass nuclei and cytoplasm from the ascogonium, which becomes more and
more vacuolate in appearance and is finally almost completely depleted. |
It is clear that the ascogonium which produces the ascogenous hyphe has
undergone no process of fertilization by male nuclei, so the development at
first sight appeared to be a truly parthenogenetic one. When, however, such
a case as the development of the accidium of Phragmidiwm violaceum
(Blackman, 5) was considered—where, in the absence of the male cell, there
is a peculiar process of fertilization by the union of a vegetative cell with the
female cell—it seemed conceivable that a reduced process of a somewhat similar
nature might be found in H. granulata also. If this were so, two possi-
bilities presented themselves ; either the ascogonium might be fertilized by the
entrance of the contents of the uppermost stalk cell or of some other vege-
tative cell, or a fusion in pairs of the nuclei of the ascogonium might take
place. As no evidence of the first possibility could be obtained, the ascogonial
contents were very closely examined at various stages of development, with
the result that the second hypothesis was found to be correct, and the female
nuclei were observed fusing in pairs in the ascogonvum.
These fusions are to be observed in ascogonia of various ages, sometimes
when the investment of the ascogonium has only just begun, but usually at
some stage between investment and the emptying of the ascogonium.
There thus appears to be no definite stage of fusion for all the nuclei
corresponding to that of Pyronema, but a gradual fusion in pairs takes
place as development of the ascogenous hyphe proceeds. The majority
of fusions were observed when the ascogonium was partly emptied of its
contents, as the nuclei are then not so deeply crowded as in earlier stages, and
the cytoplasm does not stain so deeply.
The nuclei in most stages are so close together that it is usually impossible
to distinguish from mere accidental contact the contact of nuclei which is a
358 Mr. Blackman and Miss Fraser. Sexuality and [Oct. 31,
preliminary to fusion. In a number of cases, however, nuclei were found in
pairs more or less isolated from their fellows, which were probably to be
considered as on the point of fusion. The actual fusion of nuclei seems to
take place very quickly, for such a stage as that of fig. 14 is rarely seen, but
the nucleoli apparently remain for some time separate, for the nucleus with
two nucleoli (figs. 15, 16, 17) is found more frequently. Apart from the
stages of contact, which must necessarily be impossible to distinguish with
certainty, more than 11 cases of actual fusion were observed, so there can
be no doubt that the fusion of the ascogonial nuclei in pairs is a regular
process. The size of the nuclei is not of much help in deciding whether a
given nucleus is or is not the result of fusion, as three or four different sizes
of nuclei may be observed in a single ascogonium (fig. 18). The nuclei
apparently undergo a fairly rapid growth in size, those at the centre of the
ascogonium being usually smaller, at least in the later stages, than those at
the periphery.
The number of nuclei in the ascogonium varies apparently with the size
of that structure, but in order to gain some idea of the number in an average
ascogonium the nuclei were counted in two cases in a series of sections of an
ascogonium. In the young ascogonium, of which a section is shown in fig. 7,
the number 336 was determined, while the older one of fig. 10 gave 824.
These countings are, of course, only approximate, as the nuclei are very
crowded, and lie sometimes one above the other; also in the older asco-
gonium, a small number of the nuclei had already migrated, and some of
those still remaining had, no doubt, already fused. The number produced by
the division of the original nuclei of the ascogonium might therefore be taken
as about 1000. No data were obtained as tu the number of nuclei in the
ascogonium at its first inception, but judging from the size of the organ at
that stage and from the relatively small number of nuclei in the vegetative
cells, very numerous divisions must take place. It is curious that such
divisions were never observed in the ascogonium ; it 1s probable that they
are intermittent in occurrence; possibly they take place only at night.
When the nuclei pass out into the ascogenous hyphe they show a very
distinct nucleolus and are easily defined structures (fig. 11); thus the distine-
tion of the ascogenous from the vegetative hyphe (fig. 19) among which
they ramify is rendered possible.
The ascogonium becomes finally emptied of its contents, though sometimes
a few nuclei and a little cytoplasm remain behind at the periphery. Soon
after the ascogonium becomes empty, the connection of the ascogenous hyphe
with it becomes obliterated, and these hyphz appear as independent struc-
tures. This result is no doubt brought about by the pressure of the
1905.] Development of the Ascocarp of Humaria granulata. 359
surrounding cells which leads to a slight collapse of the wall, aud so to the
obliteration of the cavity of the ascogenous hyphe at their point of origin.
Ultimately the whole ascogonium becomes obliterated, though it remains con-
spicuous as a large empty vesicle up to the time when the ascocarp first opens
(fig. 31). The stalk cells also become emptied (fig. 30), and are obliterated
somewhat earlier, so that after the early stages of opening no trace at all of
the archicarp can be observed. It may be mentioned here that the nuclei
of the uppermost stalk cell are generally more distinct than those of the
ordinary vegetative hyphe, being intermediate in structure between those
and the ascogonial nuclei (figs. 11 and 12).
In the earlier stages of development the whole of the nourishment for the
growth of the apothecium is supplied by the archicarp from its reserve of
material, the cells of the stalk supplying the branches which arise upon them,
while the ascogonium supplies the ascogenous hyphe. In the later stages,
however, a “secondary mycelium” is formed consisting of vegetative hyphe
which grow down into the substratum and absorb nourishment which is
Supplied to the vegetative hyphe of the ascocarp, and so indirectly to the
ascogenous hyphe which, after the emptying of the ascogonium, are practically
parasitic on the vegetative hyphe.
The first asci are formed very early before the outer peridium is burst
through (fig. 30); they arise on the ends of the ascogenous hyphe by the
peculiar process of the bending over of the apex and the fusion of the nuclei
in the sub-terminal cell (figs. 20 to 25), such as has been described by Harper,
Dangeard, Guillermond, and Claussen. In two cases the ascus was observed
in a terminal position as described by Maire(23) and by Guillermond (14)
for Galactinia succosa.
When the two nuclei have fused in the ascus, the fusion nucleus begins to
increase in size and to show a definite chromatin substance between nucleolus
and wall.
The division of the nucleus does not call for any particular comment, as it
is not very favourable for investigation ; the spindles are at first intranuclear
and show well-marked centrosomes with radiations, but the chromosomes are
too small to allow of an estimation of their number (figs. 26 to 28).
The method of spore formation in the ascus appears to be of the well-known
type first described by Harper, but owing to the density of the contents of
the ascus and the somewhat small size of the spores the object is not a
favourable one for the study of the details of the process (fig. 29).
The paraphyses at their first appearance form a wedge-shaped mass, which
appears to play a part in bursting open the peridium, as described by Harper
in Ascobolus.
VOL. LXXVIIL—B. 2D
360 Mr. Blackman and Miss Fraser. Sexuality and [Oct. 31,
The structure of the mature apothecium is of the ordinary type; there isa
definite parenchymatous peridium, a well-marked .hypothecium consisting of
large cells; and the paraphyses are large and club-shaped and filled with
orange granules (fig. 31).
General Considerations.
It is clear that the process of fusion in pairs of the female nuclei in the
ascogonium of Humaria granulata must be considered as a reduced sexual
process which, in the absence of the antheridium, replaces the normal fertilisa-
tion by male nuclei such as we find in Spherotheca, Erysiphe, Pyronema, and
Boudiera. It renders even more untenable the most recent view of
Dangeard* (10) that in the Ascomycetes as a whole there is no fertilisation in
the ascogonium, but the sexual process has been shifted from that structure to
the asci; for in HA. granulata we find that even in the absence of the
antheridium the process of nuclear fusion is not confined to the asci, but
there is an earlier fusion in the ascogonium, which must itself be considered
as the sexual process, although of a reduced type.
As stated earlier, the question of the occurrence of an ordinary sexual
process in some at least of the Ascomycetes must now be considered as com-
pletely settled. Future work must decide how far the members of the group
exhibit ordinary sexuality or the reduced process described above; it is possible
also that some forms are truly parthenogenetic,+ while there appears to be no
doubt that others, as the Hxoascacee, are still further reduced, the asci having
a direct vegetative origin.
_ It can hardly be denied that the process of fusion of the female nuclei in
pairs is derived by reduction from the ordinary sexual process such as we
find in Pyronema; therefore it seems best to class such a process as a
“yveduced sexual process ” (Blackman (5)), in which the male gamete has been
replaced by another female cell (nucleus), the ecidium, just as in Phragmidium
violaceum (5) the male cell is replaced by a vegetative cell.
* Kuyper (21) in a recent paper, published since these observations were complete, has
come to a conclusion somewhat similar to that of Dangeard. He has investigated
Monascus and finds there only a single nuclear fusion, and that in the ascogonium, but
without relation to the male nuclei. He considers Monascus a primitive form and that
in the other Ascomycetes the fusion has been shifted to the ascus. Different results have
been obtained by other workers on Monascus, and Kuyper’s figures are not very con-
vincing ; but if there is only a single fusion, such a fusion is obviously comparable to the
first fusion in Humaria granulata and not to the second.
+ That is with potential female gametes developing without any process of cell or
nuclear fusion. If there be a true alternation of cytologically distinct generations in the
Ascomycetes this is not likely to occur, as true and complete parthenogenesis is unknown
in plants possessing such an alternation.
1905.| Development of the Ascocarp of Humaria granulata. 361
Davis (12) has criticised such a terminology in the case of Phragmidiwm,
and objects to the use of the terms fertilization or sexual process being applied
to any union in which the fusion is not between the regular male and female
cells. He would class these irregular processes under the head of asexual
fusions.
It is true that a fusion in which the special sexual cells do not both take
part cannot, from a purely morphological point of view, be a sexual process.
When, however, it is considered that in some of these irregular fusions one of
the sexual cells actually takes part, and also that they are of very special
nature, being directly related in the phylogeny of the group to the ordinary
sexual process, in fact, replacing that process in the life history, they can
hardly be satisfactorily relegated to a class of asexual unions, where they are
herded with processes most of which have not been shown to have any
connection with true sexual fusions.* If on strict morphological grounds
these fusions are separated from true sexual processes they should obviously
be made a class apart, quite distinct from the asexual unions.t
Itis doubtful, however, whether a purely morphological test of a sexual
process (syngamy) is desirable when we consider that the process is essentially
a physiological one and that primitively it occurs between vegetative cells
(¢.g., Spirogyra, some Protozoa). Further, these irregular processes show no
characters for which a parallel cannot be found in other accepted sexual
processes ; for in the simplest cases the fusing cells are not differentiated, and
in other cases of sexuality the blood-relationship between the fusing cells
(eg., lateral conjugation in Spirogyra, sexuality in Basidiobolus and many
Phycomycetes) is apparently as close as in the process under discussion.
Since, then, these special processes in themselves have no characters which
remove them from the class of sexual unions, and since they take place
* Such as the fusion of nuclei in endosperm cells, and in cells which have been placed
under abnormal conditions, the fusion of nuclei in the ascus, the “ vegetative” cell fusions
in the Floridez, ete.
+ In the present state of our knowledge the cell and nuclear unions among plants would
seem to be best divided into four classes :—
(1) Cell-unions of an ordinary sexual nature :
(2) Reduced sexual processes as described above :
(3) Nuclear unions, such as are found in the teleutospore, basidium, and perhaps those
of the spores of the Ustilagine ; these (at least in the case of some Uredinee
and probably in the other cases) are the direct result of sexual, or reduced
sexual, processes which exhibit nuclear association without nuclear fusion :
(4) Asexual cell and nuclear unions, which are of doubtful or purely vegetative
nature.
The third class is of very special nature, and it is not satisfactory to class them, as
does Davis, with the asexual unions.
BAD We
362 Mr. Blackman and Miss Fraser. Sexuality and [Oct. 31,
at a definite point in the life-cycle, and replace in phylogeny the ordinary
sexual process, it seems proper that their relations should be exhibited in
the terminology, and they should be classed as sexual processes or fertiliza-
tions, with the addition of the term “reduced,” which indicates that one,
or both, of the regular sexual cells has been replaced by some other cell.
In the case of Phragmidium violacewm* and the “apogamous” prothallia,
we may consider that there has been a sudden return in part, or as a whole,
to the primitive condition where every vegetative (gametophytic) cell is a
gamete.
It appears, then, from the study of H. granulata that the female
ceenogamete possesses a very striking property—the capacity to fertilize itself.
It may be that in this capacity lies the explanation of the development
without male sexual organs which seems normal for a large number of
Ascomycetes ; future research can alone settle this point.
Although the sexual process to be observed in H. granulata is, of course,
morphologically reduced in relation to the normal sexual process, yet
physiologically there can be little to choose between the fusion of ascogonial
nuclei, which may be separated in descent by many divisions, and the
ordinary sexual fusion in which, as is often the case, the antheridum and
ascogonium are intimately related in origin. In fact the kinship of the
fusing nuclei may very likely be closer in Boudiera, where sexual organs
are borne in pairs on the same hypha and contain a small number of nuclei,
than in Humaria, where the number of nuclei in the ascogonium is very great.f
As has been suggested elsewhere (Blackman (6)), the majority of the
(morphologically) normal sexual fusions in the Fungi, exhibiting as they do
close-related sexual organs, are already physiologically reduced in relation
to the typical (and probably primitive) exogamic sexual process. The
morphological reduction found in the special fusions is thus only a small
step which does not affect the physiological nature. In other words,
instead of the fusion of the gametes from two gametangia borne close
together and in intimate relation on the same plant, we have the abortion
of the one and the fusion in pairs of the gametes of the other; put in this
* The case of the xcidium of another species, P. speciosum, in which neighbouring
cells fuse in pairs, described by Christman (8), and considered by him as a simple process
of conjugation of undifferentiated gametes, would seem to be much better interpreted as
a reduced sexual process, in which, in the absence of the male cells (spermatia), the
female gametes fuse in pairs, as in 4. granulata (vide 7A).
+ It is not asserted that the close kinship or otherwise of the fusing nuclei necessarily
makes any physiological difference, but that judged by this standard the processes are
essentially similar ; and it is not clear that there are any other physiological factors
which would differentiate the two processes.
1905.| Development of the Ascocarp of Humaria granulata. 363
way it is clear that there is a morphological difference, but a physiological
difference is not easily conceivable.
When one considers the apparent physiological equivalence of the
ordinary and the reduced sexual processes, the ease with which “self-
fertilization ” can be carried on in the ccenogamete, the small number of
forms in which an ordinary sexual process has been observed, and the
fairly large number which appear to have no antheridium, it seems not
improbable that the reduced sexual process will prove to be the more
common type of fertilization in the Ascomycetes.
It is obvious that the occurrence of fusions among the nuclei of the
female ccenogamete itself renders still more difficult the investigation of
the sexual cell of this type. The mere presence of a male organ and the
observations of nuclear fusions in the female cell is now not sufficient to
prove a normal fertilization ; nor even is continuity between male and female
organs, for the male nuclei may degenerate in situ, and a reduced fertilization
of the H. granulata type may take place. To prove the existence of ordinary
fertilization, evidence must be obtained for an actual migration of male
elements to the female organ.
Jt might perhaps be suggested by some that the nuclear fusion observed
by Harper in Pyronema were really fusions between female nuclei like those
in H. granulata. MHarper’s observations on the passage of the male nuclei
into the oogonium seem, however, sufficiently satisfactory to allow of this
>
supposition being put on one side.
Dangeard’s observations on Pyronema are very probably to be explained
by the supposition that he was working on a form with a functionless
antheridium. He worked with artificial cultures, while Harper used natural
ones, and it has been shown by Van Tieghem (26), in a paper which seems
to have been overlooked in the discussion, that Pyronema is very susceptible
to artificial conditions. In his cultures Van Tieghem observed forms which
were normal, forms which showed the ascogonium and antheridium reduced
in size, and lastly forms in which the antheridium was absent, but the
ascogonium developed normally. Dangeard was probably investigating a form
in which the antheridium, though still present, had already become function-
less ; in the light of the series of forms observed by Van Tieghem, one cannot
conclude with Dangeard that the antheridium is always functionless.
In such a case as Pyronema with a functionless antheridium a “ reduced
fertilization” similar to H. granulata is to be expected; such a process
would almost certainly be overlooked unless attention was specially directed
to it. The other forms lately investigated by Dangeard (10), in which either
the antheridium was absent or the male nuclei degenerated, may, perhaps,
364 Mr. Blackman and Miss Fraser. Sexuality and ([Oct. 31,
also be explained by the fact that a reduced fertilization in the ascogonium
was overlooked.
Exact data as to the nuclear behaviour of Ascobolus furfuraceus in its early
stages of ascocarp development will be of special interest. It would seem
likely that the nuclei fuse in pairs when they meet, as described by Harper (16),
in the large ascogonial cell which gives origin to the ascogenous hyphe. One
of Harper’s figures shows these nuclei in very close contact.
It is possible, also, that the parthenogenesis in other forms, in which
a coenogamete develops without the intervention of a male organ (eg., the
Mucorini and Saprolegniacez), may be explained in the same way, by
a fusion, in pairs, of the female nuclei,* and so not be a true parthenogenesis.
The fusion in the ascus still remains a most puzzling process, for which, at
present, no explanation is forthcoming. That it is not a substitute for the
ordinary sexual process, nor a nuclear fusion which has been shifted, in
descent, from the ascogonium to the ascus, as Dangeard and Kuyper believe,
is clearly shown (apart from such forms as Pyronema, Boudiera, etc.) by
H. granulata, where, even in the absence of the antheridium, the fusion in
the ascus is preceded by a fusion in the ascogonium. On the other hand,
the curious simultaneous division of the two nuclei at the time of ascus
formation—whether the ascogenous hypha bends over at the apex or whether
it remains straight, as in Galactinia succosa—seems only to be explained as a
method of ensuring that the fusing nuclei are separated in descent by at
least one division. Now, such a separation, in descent, of the fusing nuclei
is, as far as we know, an attribute of sexual fusions alone (though in many
fusions which are accepted as sexual, the degree of relationship is very close).
We have thus two closely related fusions, one of which is obviously a sexual
fusion, while the other, in one character at least, partakes of a sexual nature.
A satisfactory solution of the difficulty of the dual fusions can hardly be
expected till we know the number of chromosomes throughout the life-history
of some ascomycete.
That there is a definite alternation of generations in the life-history of
Ascomycetes which possess an ascogonium seems very probable. The
ordinary vegetative mycelium would appear to be the gametophyte, which
bears the ascogonium, and antheridium if present, while the products
of fertilization, the ascogenous hyphe (which are parasitic on the
gametophyte) and the asci, represent the sporophyte. The countings of
chromosomes are, however, too few and too unsatisfactory to allow of
* Kuyper (21) has independently made a similar suggestion in the case of the
Saprolegniacez, and has even suggested that the figures of Davis (11) on egg development
in Saprolegnia support the view of a nuclear fusion.
1905.| Development of the Ascocarp of Humaria granulata. 365
a decision. as to the cytological distinction of the two generations. It
would seem, also, that there must be two reductions, as there are two
fusions. The three divisions in the ascus might be expected to show at
least one reduction, but Harper(17) is of the opinion that the number of
chromosomes remains unaltered during these divisions. Of course, it is
possible that the second fusion is of a peculiar nature and does not lead to
a doubling of the chromosomes. What is obviously necessary is the dis-
covery of a sexual ascomycete with a small number of distinct chromosomes
in its nucleus, so that the number can be observed throughout the life-
history.*
De Bary uses the term archicarp as practically synonymous with
ascogonium. It seems much more satisfactory to use the term archicarp
for the whole fertile branch, apart from the antheridium, and to confine
the term ascogonium to that part of the archicarp the contents of which
take part in the formation of ascogenous hyphe, 7. the reproductive
cell or cells which contain the female nuclei. It is in this sense that the
terms have been used in the body of the paper. Used in this sense the
term ascogonium is not necessarily confined to the cell or cells actually
giving origin to the ascogenous hyphe. In Ascobolus furfuraceus, for
example, the whole curved fertile branch, or scolecite, is the archicarp;
the central part would be the ascogonium, which is divided into a number
of cells by a series of perforate septa, as Harper (16) has shown; only one
of the cells of the ascogonium, however, actually gives origin to the ascogenous
hyphe, though the contents of all the ascogonial cells pass into this special
cell and so into the ascogenous hyphe. In Melanospora parasitica, from the
observations of Kihlmann, the ascogonium is represented by one or two cells
of the archicarp, though the cytological details are not known. In Pyronema,
Humaria, Spherotheca, and Erysiphe the ascogonium is a single cell, and
naturally gives origin to one or more ascogenous hyphe. In Collema the
archicarp consists of a few small sterile cells at the base, then comes the
ascogonium, which is multicellular, and above is the multicellular trichogyne ;
all the cells of the ascogonium appear to give origin to ascogenous hyphe.
* The view that there are two reduction processes is also put forward by Harper in a
very important paper (“Sexual Reproduction and the Organization of the Nucleus in
certain Mildews,”) received while the present paper was passing through the press.
366
Mr. Blackman and Miss Fraser. Sexuality and [Oct. 31,
LIST OF PAPERS.
Barker, B. T. P. “The Morphology and Development of the Ascocarp in
Monascus,” ‘ Annals of Botany,’ vol. 17, 1903, p. 167.
Barker, B. T. P. “Further Observations on the Ascocarp of Ryparobius,” Leaflet,
Brit. Assoc. Meeting, 1904.
Baur, E. “ Die Anlage und Entwickelung einiger Flechtenapothecien,” ‘ Flora,’
vol. 88, 1901, p. 319.
Baur, E. “Untersuchungen iiber die Entwickelungsgeschichte der Flechten-
apothecien,” ‘ Bot. Zeit.,” vol. 62, 1904, p. 21.
Blackman, V. H. “On the Fertilization, Alternation of Generations and General
Cytology of the Uredinez,” ‘ Annals of Botany,’ vol. 18, 1904, p. 323.
Blackman, V. H. “On the Relation between Fertilization, ‘Apogamy’ and
‘ Parthenogenesis,’” ‘ New Phytologist, vol. 3, 1904, p. 149.
Blackman, V. H., and Fraser, H.C. I. “ Fertilization in Sphzrotheca,” ‘ Annals of
Botany,’ vol. 19, 1905, p. 567.
. Blackman, V. H., and Fraser, H.C.1I. “Further Studies on the Sexuality of the
Uredinez” (zbzd., vol. 20, 1906, pt. 1).
Christman, A. H. “Sexual Reproduction in the Rusts,” ‘Botan. Gazette,’ vol. 39,
1905, p. 267.
. Claussen, P. “Zur Entwickelungsgeschichte der Ascomyceten. Boudiera,” ‘ Bot.
Zeit.,’ vol. 63, 1905, p. 1.
Dale, E. “Observations on the Gymnoascaceze,” ‘Annals of Botany,’ vol. 17, 1903,
p- 571.
Dangeard, P. A. “Recherches sur le Développement du Périthéce chez les
Ascomycetes,” ‘Le Botaniste,’ 9e série, 1904, p. 59.
. Darbishire,O. V. “ Die Apothecienentwickelung von Physcia pulverulenta,” * Jahrb.
f. wiss. Botanik,’ vol. 34, 1908, p. 329.
Davis, B. M. “ Oogenesis in Saprolegnia,” ‘ Botan. Gazette,’ vol. 35, 1903, p. 233.
Davis, B. M. “Studies on the Plant Cell,” ‘The American Naturalist, vol. 35,
1905, p. 217.
Fiinfstuck, M. “ Beitrige zur Entwickelungsgeschichte der Lichenen,” ‘Jahrb. d.
K. Botan. Gart. zu Berlin,’ vol. 3, 1884, p. 155.
Guilliermond, A. ‘Contrib. 4 étude de la Formation des Asques,” ‘Rev. Gén. de
Botanique,’ vol. 16, 1904, pp. 49 and 129.
Harper, R. A. “Die Entwickelung des Peritheciums bei Spherotheca Castagnei,”
‘Ber. d. d. Bot. Geo.,’ vol. 13, 1895, p. 475.
Harper, R. A. ‘Ueber das Verhalten der Kerne bei der Fruchtentwickelung
einiger Ascomyceten,” ‘ Jahrb. f. wiss. Botanik,’ vol. 29, 1596, p. 655.
Harper, R. A. “Sexual Reproduction in Pyronema confluens, etc.,” ‘Annals of
Botany,’ vol. 14, 1900, p. 321.
‘Holtermann, ©. “Mykologische Untersuchungen aus den Tropen,” Berlin, 1898.
Janezewski, E. ‘Morphologie des Ascobolus furfuraceus,” ‘ Botan. Zeit.,’ vol. 29
1871, p. 257.
Kihlmann, O. “Zur Entwickelungsgeschichte der Ascomyceten,” ‘ Acta Soc. Se.
Fennice,’ vol. 14, 1885, p. 309.
Kuyper, H. P. “Die Perithecien-Entwicklung von Monascus purpureus, Went.
usw.,” ‘ Annales Mycologici,’ vol. 3, 1905, p. 32.
Lindau, G. “ Die natiirlichen Pflanzenfamilien,” Teil 1, Abt. 1, 1897, p. 323.
Maire, R. “Recherches cytologiques sur les Ascomycétes,” ‘Annales Mycologici,
vol. 3, 1905, p. 123.
1905.] Development of the Ascocarp of Humaria granulata. 367
24. Oltmanns, F. ‘“ Ueber die Entwickelung der Perithecien in der Gattung
Cheetomium,” ‘ Bot. Zeit.,’ vol. 45, 1887, p. 192.
25. Woronin, M. “Zur Entwicklungsgeschichte des Ascobolus pulcherrimus, Cr. usw.,”
‘ Beitr. zur Morphol. u. Physiol. der Pilze, Zweite Reihe,’ 1866, p. 1.
26. Van Tieghem, Ph. “Culture et développement du Pyronema confluens,” ‘ Bull. de
la Soc. Bot. de France,’ vol. 31, 1884, p. 35.
EXPLANATION OF PLATES.
PLATE 13.
Fig. 1.—Portion of mycelial hypha showing the nuclei, and deeply-staining granules on
the transverse wall. x 1900.
Fics. 24 and 28.—Two young archicarps growing up from the general mycelium : fresh
preparations. x 430.
Fic. 3.—Slightly older archicarp in section. x 430.
Fie. 4.—Archicarp showing the vegetative hyphze beginning to grow out from the cells
beneath the ascogonium. The granules on the wall are clearly visible. x 620.
Fic. 5.—Young ascocarp in which the ascogonium and the sub-terminal cells of the
archicarp are covered in by the vegetative hyphe. x 430.
Fig. 6.—Section of young ascogonium showing ascogonial nuclei. x 1050.
Fic. 7.—Section of slightly older ascogonium. x 1050.
Fic. 8.—Section of ascogonium and basal cell of archicarp which have just become
covered in by the vegetative hyphe. The vegetative as well as ascogonial
nuclei are clearly visible. x 1050.
Puate 14,
Fic. 9.—Section of ascocarp of about the age shown in fig. 5. The granules in the
ascogonium and the partial emptying of the stalk cells are to be clearly seen.
x 430.
Fie. 10.—Section of upper part of young ascocarp showing ascogonium partly filled with
nuclei and “basal cell” below. Three ascogenous hyphe can be traced
throughout their whole length, while portions of others are visible among the
vegetative plectenchyma. Nuclei are to be faintly distinguished in the “ basal
cell” and some of the vegetative cells. x 1050.
Fic. 11.—Section of somewhat older ascocarp showing the ascogonium and three stalk
cells. The majority of the nuclei have already migrated from the ascogonium.
x 620.
Fic. 12.—Section showing the nuclei in the basal cell and the cells immediately
surrounding. x 1050.
Fic. 13.—Section through the lower part of the ascogonium and the upper part of the
basal cell showing the curious granular masses on the transverse wall. x 1010.
Fic. 14.—Two female nuclei of the ascogonium in the process of fusion. x 2700.
Fic. 15.—A group of ascogonial nuclei with a fusion-nucleus showing nucleoli in act of
fusion. x 2700.
Fic. 16.—A group of nuclei of ascogonium with a large-fusion nucleus with two nucleoli.
x 2700.
368 Sexuality, etc., of the Ascocarp of Humaria granulata.
PLATE 15.
Fie. 17.—A group of nuclei from an ascogonium showing one fusion-nucleus with two
nucleoli, another with nucleoli which are just fusing. x 2700.
Fic. 18.—Group of ascogonial nuclei of different sizes. x 2700.
Fic. 19.—Ascogenous hyphe and vegetative hyphe showing the distinction of the nuclei.
x 1900.
Fics. 20—25.—Stages in the development of the ascus at ends of the ascogenous hyphe.
Fies. 26—28.—Three stages of the first nuclear division in the ascus. x 1900.
Fic, 29.—Spore formation in the ascus. x 1900.
Fic. 30.—Section of young ascocarp showing the wedge of paraphyses bursting through
the peridium. The nearly empty ascogonium and stalk cells are visible.
x 175.
Fic. 31.—Section through slightly older ascocarp when the peridium has been burst
through completely, the hymenial layer definitely arranged, and a certain
number of spores formed. The empty ascogonium is still visible, but the rest
of the archicarp has disappeared. x60.
[se
Roy. Soc. Proc. B. vol. 77 Pl
*
vy & PTASEr
Highley, imp.
ith.
. Highley, del.et1
eat,
ae
TLOV. SOG FOC DiVOUL LLL L4
P. Highley, del. et lith. Highley, imp.
P Highley, del. et lith. Highley, imp.
369
A Study of the Mechanism of Carbon Assimilation in Green
Plants.
By Francis L. UsHer and J. H. PriEstLEy, B.Sc., Lecturer in Botany at
University College, Bristol.
(Communicated by Professor M. W. Travers, F.R.S. Received December 16,
1905,—Read January 18, 1906.)
(From the Chemical and Botanical Departments, University College, Bristol.)
introduction.—The investigation to be described in this paper has had for
its object the elucidation of certain problems concerning the nature of the
first stages in the assimilation of carbon from carbon dioxide by the green
parts of plants; and although far from complete, it has been thought
advisable to publish the results already obtained, inasmuch as the weather is
likely to hinder the experimental work for some time to come.
In 1870 Baeyer put forward the hypothesis that formic aldehyde is the
first product of the decomposition of carbon dioxide in the plant. This
suggestion received some support from Bokorny,* who proved in 1891 that
starch was formed in the dark by the green filaments of Spirogyra when
immersed in a solution of sodium oxymethyl-sulphonate of 0:1 to 1 per cent.
strength.
Bokorny’s experiments are possibly open to the objection that formaldehyde
condenses very readily to non-poisonous carbohydrates in presence of sulphites
or bisulphites, and it has been shown by Laurent and Actont that starch is
formed in the dark from most sugars.
Quite recently, Bouilhac and Trébouxt have succeeded in growing plants
_ In a very dilute solution of pure formaldehyde. Tréboux has found that
Hlodea forms starch in the dark from a 0:001-per-cent. solution of formal-
dehyde, and Bouilhac has shown that this is also the case with Sinapis alba
and some Algw. Their experiments bring out in a striking manner the
intensely poisonous nature of even very dilute solutions of formaldehyde.
Evidence of this kind, however, is quite indirect, and on this account greater
importance attaches to the results obtained by Bach,§ who for the first time
demonstrated the decomposition of carbon dioxide by light outside the plant.
He showed that by passing pure carbon dioxide through a 1:5-per-cent.
* ‘Berichte,’ 1891, vol. 24, p. 103.
+ ‘Roy. Soc. Proc.,’ 1890, vol. 47, p. 150.
{ ‘Flora,’ 1903, p. 73.
§ ‘Comptes Rendus,’ 1893, vol. 116, p. 1145.
370 Messrs. F. L. Usher and J. H. Priestley. _[Dec. 16,
solution of uranium acetate exposed to sunlight in a glass apparatus, a pre-
cipitate consisting of a mixture of uranium peroxide with lower oxides was
formed, and that the solution contained formaldehyde. Bach regarded the
uranium acetate solution as playing the part of a chemical and an optical
sensitiser, and considered the decomposition of the carbon dioxide to result
primarily in the production of hydrogen peroxide and formaldehyde.
Decomposition of Carbon Dioxide Outside the Plant.—The experiments of
Bach have been repeated and confirmed, both as to the production of peroxide
and formaldehyde.
The amount of decomposition obtained in three weeks in bright weather
was extremely small, and this appears to us to be explained by the fact that
(1) as a chemical sensitiser uranium acetate is far inferior to that which exists.
in a green plant, inasmuch as the separated oxygen (in whatever form it may
exist) is not entirely removed from the sphere of action, as in the case of
the plant, but remains as a fairly insoluble peroxide which undergoes a rever-
sible change with the other product, namely, formaldehyde ; (2) as an optical
sensitiser uranium acetate is inferior to chlorophyll to the extent that it .
possesses no absorption at all in the red, and only two faint bands between
F and G.
In view of the extreme slowness of the reaction under these conditions,
experiments were made with very large concentrations of carbon dioxide.
Tubes of Jena glass, 40 cm. long and 8 to 10 mm. bore, were about three-
quarters filled with 1:5 per cent. uranium acetate solution, and cooled in liquid
air while some carbon dioxide was passed in. They were then sealed, and
suspended outside a south window in bright sunshine. Within 15 minutes of
warming up to the air temperature, a precipitate began to form, and in
24 hours the reaction was complete. The tubes when opened were found to
contain uranium peroxide and formic acid, but no formaldehyde. The formic
acid was obtained by distillation of the filtrate from the peroxide, and was
characterised by (1) reduction of silver nitrate, (2) reduction of Fehling’s.
solution, and (3) properties of lead salt.
Thus with very large concentrations of carbon dioxide, formic acid, and not.
formaldehyde, results.
These experiments are open to the objection that since uranium acetate 1s:
to a considerable extent hydrolysed in solution, the formaldehyde in one case
and the formic acid in the other may possibly have been derived from the
acetic acid present.
It has been found that uranium sulphate in a 2-per-cent. solution functions.
in the same way as the acetate. An experiment with the sulphate, conducted
in the usual way, 2.¢., bubbling carbon dioxide through the solution, which
1905.| Mechanism of Carbon Assimilation in Green Plants. 371
lasted over three weeks in very dull weather, gave uranium peroxide and
formic acid. The different results obtained here from those in the case of
similar experiments with the acetate, may be due to the “reduction potential”
falling below the limit required for the completion of the second stage of the
decomposition.
In all the foregoing experiments, except in the case of the liquid carbon
dioxide tubes, blank experiments were simultaneously performed, (1) with
uranium solution and carbon dioxide in the dark, and (2) with carbon dioxide
free solution in the light. In neither case was any precipitate formed.
Decomposition of Carbon Diowide in the Plant—I{ a similar reaction,
resulting in the formation of formaldehyde and a peroxide, takes place
in the first stage of the absorption of carbon dioxide by the plant, it
is obvious that both the initial products of decomposition must undergo
a rapid change.
On account of its intensely poisonous nature, formaldehyde must be very
rapidly converted into some physiologically inert substance ; and the peroxide
must be decomposed with evolution of gaseous oxygen, a process which
follows exposure to light by an interval of one or two seconds.
The problem, then, is to ascertain the process by which oxygen is dis-
engaged; to show the actual presence of formaldehyde localised in the
neighbourhood of the chloroplasts; and to trace the steps by which the
formaldehyde is polymerised.
The Mechanism of the Evolution of Oxygen from the Green Plant.—In the
experiments relating to the decomposition of carbon dioxide outside the
plant, no evolution of oxygen gas is ever observed; it remains in the system
as a peroxide.
There have been conflicting statements with regard to the presence of
hydrogen peroxide in plants, but even if traces are to be found, there is no
evidence that it 1s a product of decomposition of carbon dioxide. It has
indeed been shown that several organic substances, notably the organic acids,
é.g., oxalic, give rise to hydrogen peroxide on exposure to light, and such
substances as these are of common occurrence in the leaves of plants.
If, however, hydrogen peroxide is one of the first products of the photolysis
of carbon dioxide, we are more directly concerned with the elimination of
oxygen in the gaseous form than with the detection of the peroxide.
Hitherto those writers who have recognised the difficulty at all have
suggested some method of reduction, which, of course, leads back to the
starting point. It appeared much more probable that this step in the process
was brought about by a catalyst, probably an enzyme. To test this, some
Elodea was immersed in a dilute solution of hydrogen peroxide. An
372 Messrs, F. L. Usher and J. H. Priestley. _[Dee. 16,
immediate and rapid decomposition set in, and a gas was freely evolved,
which was found to be oxygen. The action proceeded as rapidly in the dark
as in the light.
The following experiments were performed with the object of ascertaining
the nature of the catalyst :—
(a) A plant was immersed in boiling water for 50 seconds and was sub-
sequently found to be without action on hydrogen peroxide.
(b) After treatment with dilute solutions of iodine, mercuric chloride, and
hydrogen sulphide, no action took place.
(c) Some Elodea was suspended in air charged with chloroform vapour for
two hours in order to kill the protoplasm, and was then allowed to “ recover ”
for a similar period. Rapid disengagement of oxygen took place.
(d) After immersion in very dilute formaldehyde solution, hydrogen
peroxide was not decomposed.
These experiments seem to point to the existence of a catalysing enzyme.
Several attempts to extract it by simple maceration with water or salt
solution failed, and we were also unable to extract it after powdering leaves
in liquid air. Following a suggestion of Dr. Horace Brown, we ultimately
succeeded in obtaining it by previously drying a quantity of Hlodea, and
subsequently digesting with water at 30° for 48 hours. The enzyme was
precipitated by an excess of absolute alcohol and dried.
By this process it is obtained as,a light brown powder, containing diastase,
whose aqueous solution energetically decomposes hydrogen peroxide, whereas
ordinary malt diastase does not. Whether the enzyme is one already known,
or whether it is secreted specially for the purpose of catalysing. hydrogen
peroxide, we cannot as yet say.
On mounting a leaf of Hlodea in very dilute hydrogen peroxide, and
examining microscopically under a high power, bubbles of gas were seen
to emerge from the chloroplasts only, an observation which shows the strict
localization of this enzyme to the seat of the photosynthetic process.
In regard to the distribution of this enzyme, we have examined the
foliage leaves of plants belonging to 46 Natural Orders and representative
of the Vascular Cryptogams and all the main groups of the Phanerogams,
and have found the power of catalysing hydrogen peroxide in every case,
though the energy of the decomposition varies considerably in different
groups. It also occurs in etiolated leaves and in potato tubers, and, in fact,
appears to be associated with amyloplasts, whether possessing chlorophyll
or not.
The Production of Formaldehyde and the Manner of its Removal_—tit has
been found in the case of Spirogyra that starch appears in a previously
1905.| Mechanism of Carbon Assimilation in Green Plants, 373
starchless filament within three minutes of exposure to light, and it is
probable that some sort of carbohydrate is formed much sooner than this,
for it has been shown by Brown and Morris* that starch is probably§not
elaborated within the cell until the supply of nutriment is in excess of the
cell requirements.
It would therefore seem as though the arrangement which exists in the
plant for the removal of formaldehyde is at least as efficient as any external
arrangement we can make to remove it in a different way, without at the
same time killing the plant, and thus eliminating one of the essential
factors, namely, the vitality of the protoplasm.
For this reason it is useless to look for formaldehyde in healthy
assimilating leaves. It is well known that certain chemical substances
possess the property of condensing formaldehyde to various carbohydrates,
chiefly formose, a-acrose, and methylenitan. It has been found by Loewt
that such condensing agents are chiefly metallic oxides and acid sulphites,
substances not likely to occur in plants.
Moreover, condensation by these bodies is a comparatively slow process,
and quite inefficient when applied to the requirements of a plant.
Nevertheless, if the condensation in the plant were due to some chemical
agent stored in the neighbourhood of the chloroplast, it should still be
capable of taking place when the protoplasm of the leaf is killed and its
enzymes destroyed.
Some healthy green sprigs of Hlodea were immersed in boiling water for
30 seconds, in order to kill the protoplasm and destroy the enzymes. They
were then placed in water saturated with carbon dioxide and exposed to
sunlight. In the course of a few hours the deep green colour of the leaves
had been completely bleached, and on immersing the bleached sprigs in
a solution of rosaniline decolourised with sulphurous acid, a red colour was
developed.
The original green material when treated in this way exhibited no
colouration. There was, therefore, some substance of an aldehydic nature
present in the killed and bleached leaves which was absent in those which
were alive. The sequence of events in this experiment may be described
as follows :—Photolysis of carbon dioxide begins in the normal way, giving
rise to hydrogen peroxide and formaldehyde. The enzymes having been
destroyed, the hydrogen peroxide, instead of being catalysed in the usual
manner, oxidises the chlorophyll to a colourless substance, at which point
the reaction necessarily comes to an end. Meanwhile a quantity of
* ©J.C.S.,’ 1893, ‘ Trans.,’ p. 632.
+ ‘Berichte,’ 1888, p. 271.
374 Messrs. F. L. Usher and J. H. Priestley. [Dee. 16,
formaldehyde, equivalent to the hydrogen peroxide required to destroy the
chlorophyll, accumulates, and thenceforward the reaction is strictly
reversible. /
The following experiments were performed to settle the points involved in
this explanation :—It was in the first place necessary to show whether the
colouration referred to above was due to formaldehyde. For this purpose
some leaves, killed and bleached in carbon dioxide solution as described,
were soaked for 12 hours in aniline water, and were then examined micro-
séopically under a high power. Some leaves which had been killed and
simply decolourised with hydrogen peroxide were treated in the same way.
In the first case the decolourised chloroplasts were observed to be the
centres of clusters of well-defined crystals, identical in appearance with those
of methylene aniline, artificially prepared from aniline water and form-
aldehyde. They were soluble in dilute mineral acids and also in warm
alcohol, from which they crystallised in the cell on cooling. The leaves
artificially decolourised with hydrogen peroxide showed no crystals.
An attempt was then made to obtain the formaldehyde outside the
plant. For this purpose a large quantity of Ulva and Enteromorpha was
killed and bleached in carbon dioxide solution, and subjected to steam
distillation. The distillate was divided into two parts. To the larger of
these was added some aniline water.
A white precipitate was formed after some time, which was collected,
and heated side by side with a comparison tube containing methylene aniline.
It melted, not quite sharply, three or four degrees below the pure artificially
prepared substance. The other portion of the distillate was evaporated with
-ammonia on the water-bath, and the residue dissolved in water and treated
with bromine water, gave the characteristic tetra-brom derivative of
hexamethylene-tetramine.
Hence, leaves in which both protoplasm and enzymes have been killed, when
placed under conditions favourable for assimilation, develop formaldehyde,
until the photolytic process is brought to an end by the destruction of the
chlorophyll.
It was next necessary to determine whether the condensation of the
formaldehyde is due to an enzyme secreted by the chloroplast, or whether
the protoplasm of the granule itself effected it. Some H/odea was suspended
in air charged with chloroform vapour for two hours, by which means the
protoplasm was killed without affecting the enzymes. It was then exposed
to sunlight in saturated carbon dioxide solution. In a few hours the
chlorophyll became bleached, and formaldehyde was subsequently found in
the plant.
1905.| Mechanism of Carbon Assimilation in Green Plants. 375
It follows from this that the protoplasm of the chloroplast is the con-
densing agent. The bleaching of the chlorophyll in this case is due to the
fact that the enzyme, though unharmed at the commencement of the
experiment, quickly becomes poisoned by the accumulating formaldehyde.
In this last experiment the limit to the accumulation of formaldehyde in a
plant is realised, since there is formed an amount equivalent to that amount:
of hydrogen peroxide which is catalysed before the enzyme ceases to act
together with an amount equivalent to the hydrogen peroxide required to
destroy the chlorophyll.
That a certain amount of hydrogen peroxide is catalysed when the
protoplasm only is dead was shown in the following manner. Approxi-
mately equal quantities of Hlodea were taken, one of which (A) was killed
by immersion in boiling water, another (B) was suspended for two hours in
air saturated with chloroform vapour to kill the protoplasm and not the
enzymes, while the third (C) served as a control.
All three were placed in carbon dioxide solution under funnels with
inverted test-tubes, and exposed to artificial light for 12 hours. From A
there was no evolution of oxygen, from B 0°3 e.c. were given off, and from
C 28 c.c. In the case of B, after exposure to light, no catalytic action on
hydrogen peroxide could be observed, while previously vigorous decomposition
had taken place.
Summary.
1. The photolysis of carbon dioxide may take place outside the plant in
absence of chlorophyll, provided one of the products is removed.
2. The normal products of the photolysis are hydrogen peroxide and
formaldehyde, though under certain conditions formic acid may be
formed.
3. In the plant the decomposition of the hydrogen peroxide is provided
for by a catalysing enzyme of general occurrence.
4. The condensation of the formaldehyde is dependent on the healthy
condition of the protoplasm.
There are therefore three factors essential to photosynthesis from carbon
dioxide and water in the plant, they are (i) vitality of the protoplasm,
(11) presence of a catalysing enzyme, and (iii) presence of chlorophyll. If
any one of these factors be interfered with, the process of photosynthesis
ultimately comes to an end, through the destruction of the optical seusitiser,
chlorophyll.
LXXVII.—B, 25
376 Mechanism of Carbon Assimilation in Green Plants.
The relations between the various factors in this process may be diagram-
matically expressed thus :——
Carbon dioxide + Water
we
—_ =
| [f not removed, destroys|= CHLOROPHYLL
h
if =
Hydrogen peroxide + Formaldehyde
[If not removed, poisons |
x
ENZYME LivIn@ PROTOPLASM
Oxygen Carbohydrates
In conclusion, we wish to express our indebtedness to Dr. Travers, and to
Dr. Horace Brown, for their valuable suggestions and help in the course of
this research.
377
A Biometrical Study of Conjugation in Paramecium.
By Raymonp Pearz, Ph.D.
(Communicated by Professor Karl Pearson, F.R.S. Received November 15,—
Read December 7, 1905.)
(Abstract.)
1. A study of variation and correlation in conjugating and non-conjugating
specimens of the common ciliate infusorian, Paramecium caudatum, was
undertaken for the purpose of obtaining answers to the following questions :—
a. Is the portion of the Paramecium population which is in a state of
conjugation at a given time differentiated in respect of type or variability
or both from the non-conjugating portion of the population living in the same
culture at the same time ?
b. Is there any tendency for like to pair with like (“ homogamy ”) in the
conjugation of Parameecium, and if so, how strong is this tendency ?
The material on which this paper is based is comprised in eight series,
taken from three different cultures at different times, and includes altogether
the measurements of 1894 individual Paramcia. The characters studied
were length and greatest breadth of the body, length-breadth index, and
the difference in Jength between the two individuals of a pair of conjugants.
In the measuring conjugant pairs were taken quite at random, and then in
each case the two undistorted non-conjugant individuals which were lying
nearest in the field of view of the microscope to the conjugant pair were
measured. This procedure was followed to avoid any sub-conscious bias in
choosing non-conjugants.
The cultural history of the different series may be summarised as
follows :—
Series A, C, D, and #.—These series all came from a single culture in the
Zoologisches Institut, at Leipzig. This culture was set with dry hay and
pond water, July 25,1905. The dates of collection and measurement and
number of individuals in each series are given in the table on p. 378.
Series B.—This series came from another culture at Leipzig set in the same
manner as the one just mentioned. Conjugants were found on August 22,
but in very small numbers. On the next day only two pairs of conjugants
were found, and after that none at all. So that, all told, Series B included
only 12 pairs of conjugants and 24 non-conjugants.
Series AA, Fy, and Fy.—These series included only conjugants. ‘They were
378 Dr. R. Pearl. A Biometrical [Nov. 15,
measured from material in the Zoological Laboratory of the University of
Michigan, collected by Professor D. C. Worcester. They all came from a
single culture set with decaying plant material and pond water. Series AA
includes 200 pairs of conjugants, Series Fy 70 pairs, and Series Fy, 77 pairs.
Number Number of
Series. Dates of measurement. of conjugants | non-conjugants
measured. measured.
JN se5008 August 15, p.m.—August 18, noon, inclusive ...... 105 pairs 210
Cae ce: » 24, AM.— ,, 26, P.M., se asaBeo ioxl Fs, 202
DE enn Fyatt BOLI dae seescbaceters soutcotseretnesetedmadectvasioas UGS 32
I Boc0e aSeptemiberiGicsce-0,-sercceaceceteaeaaysties-eesees eer eee eee _ 132
For further details regarding the measurements, culture histories, etc., the
complete paper must be consulted.
2. An examination of the variation constants shows that Paramecium is
relatively slightly more variable in breadth than in length of body, though
the difference is not large. For the variation in length the coefficients of
variability for different series (including several other long series besides
those collected in this work) are found to cluster well together about a value
of 8 to 9 per cent. This is a much lower value than has been found by
other workers{ for variation in similar size characters in organisms with firm
exoskeletons. In the characters studied Paramecium follows the same
general laws which have been shown to hold for continuous variation in
higher forms.
3. It was found that conjugants are markedly differentiated from non-
conjugants living in the same culture in both type and variability. This
differentiation includes all the characters studied. An idea of its extent
and direction may be gained by an examination of the following table.
In it are given (a) the absolute differences between conjugants and non-
conjugants in respect to the character and constant designated; (?) the
probable errors of these absolute differences; (¢) the relative differences
defined as the percentage which the absolute difference is of the non-
conjugant constant in each case. The absolute differences are taken as
positive when the non-conjugant constant is greater. Only two series
(A and C) are taken here as illustrations. In the complete paper similar
data for other series are given in detail.
* Series D and B (vide infra) include only a few individuals, because at the time they
were collected no more conjugants were to be found in the cultures from which they
came.
+ For detailed references, see complete paper.
1905. | Study of Conjugation in Paramecium. 379
Table I.—Differentiation of Conjugants from Non-conjugants.
| Ore Relative differ-
] Absolute difference eGR
Series. | Character. Constant. berween Pon sboningants non-conjugants
JOBE. and conjugants.
Per cent.
A Length ...... NIGER. ocecboedoadanshoesben 21 °833 +0°893 micron 11°5
my) Pe REE er Standard deviation 4,337 +0°631 53 27:°9
0 mp. toBd400 Coefficient of variation 1°517 +0349 per cent. 18 °5
a Breadth...... Means vasieas oisene scene 8°456 +0°335 micron 16:0
5 TRE aes Standard deviation ... 1°700 +0°237 3 28 -96
5 Toni tesa Coefficient of variation} 1°714 +0484 per cent. 15 ‘4
, Iniclexveasessse Mieaniens tacecucsecsaactoes 1510 +0°171 2 54
y) OA Uae aeaiiel Standard deviation ...| —0°195*+0°121 a ate
s Length and} Coefficient of correla- 0 °3107 +0 °0526 52°7
breadth tion
C Length ...... Meam \scaicceacieecoonaorss 33 341 +1°098 micron 15°9
Ss Regence Standard deviation ...) 6°005 +0°777 5 31 “4
Fh Fi sede Coefficient of variation 1°684 +0°398 per cent. 18 °5
rf Breadth...... MC aM s sriseciesnesanessteaes 11 °050 +0324 micron 20 ‘4
) opi) cconaon Standard deviation ...) 2°491 +0°229 55 42 *2
90) Ot ecadag Coefficient of variation 2°984 +0°456 per cent. 27 “A
p TeaVelOs |," 86:0 Not
acid : | determined
|
Table II.
| de \\valyly? Zeist 8 | 4.
ee es ee S
PALCOHOM ae ececsccce tae seanencssemenien 1°34 1-28 1-43 1:01
| AGE Sa5osanconssqesooas0de300~ 0°31 0°25 0 52 1°13
| Lacticacid .-.-....... 0... cose one 0°33 0°28 0°55 91!
|eStccinic acid’ 20)... ..c-eeneceseseees 0-15 0°19 0-27 0°32
| Formic acid ...........2...:.0.seeceeee 0 04 0-02 0:07 0-00
Carbon) dioxides.s---.2--00--ees-ee-ess 1°60 — 1°44 0°74
otal urs. ee 3°77 | o- | 4°28 | 511
Hydrogen, atoms per molecule ...| 1°33 | — | 1-50 | Wey
It will be observed that the ratio of hydrogen to carbon dioxide by volume
is about 0°5 to 1, whilst these gases are produced by JB. colt communis
in approximately equal volumes. Theobald Smith,* using an ordinary
fermentation tube, gives the characteristic ratio for B. coli communis as
H2/CO2 = 2:1 and for B. lactis aerogenes H2/CO2=1:1. This difference
is due to the solubility of the carbon dioxide in the liquid medium, and
it must be remembered that while Smith’s ratios give a perfectly satisfactory
working test for the discrimination of the organisms, they do not represent
the actual volumes or ratios of the gases produced.
Further examination of the fermentation products revealed the fact that
* Loe. cit.
1905.] __ B. lactis aerogenes on Glucose and Mannitol. 401
no other acids had been formed, and search was therefore made for com-
pounds of a different type. It was previously suggested that the deficiency
of carbon observed in the fermentation produced by L. coli communis,
amounting to only 0°25 to 0-9 of an atomic proportion of carbon, might
possibly be due to the presence of reduction products of sugar, and com-
pounds of this kind were therefore sought.
It was found that when the neutral liquid, containing the products of
fermentation along with peptone, was evaporated to dryness at 55° under
diminished pressure and extracted with alcohol, a solution was obtained
which yielded on fractionation a colourless liquid boiling at 181° to
183° (corr.) at 760 mm. pressure. The yield was very small, only amounting
to about 1 gramme per litre of medium containing 20 grammes of glucose,
but it was found possible to increase the yield by employing a medium
containing 5 per cent. of glucose, and in this way 8 grammes of the new
substance, containing 52°8 per cent. of carbon, were obtained per litre of
medium contaiming 50 grammes of sugar. This only accounts for about
two-thirds of the missing carbon, and a rough estimate of the amount
lost during the process of distillation and extraction was, therefore, made
by dissolving 8 grammes of the material in 500 c.c. of a medium containing
5 grammes of Witte peptone, 6 grammes of calcium lactate and 65 grammes
of alcohol and then extracting it in the manner described above. Only
a2 grammes were recovered, the loss per 500 ec. being therefore about
2°$ grammes and the loss per litre about 56 grammes. This brings the
total amount produced from 50 grammes of glucose to about 13°6 grammes,
slightly in excess of that required. It is hoped that the actual yield may
be increased by a careful fractionation of the fermentation products.
The new product is apparently a mixture, and it has not yet been found
possible to separate and identify all the components, so that the following
must be taken as only a preliminary account of the substance.
It boils at 181° to 183° (corr.), and solidifies in the cold to a transparent
mass which melts indefinitely at about 28°. It is optically active, the value
for [¢]p for different preparations varying from 0-46 to 0-71. The com-
position of the substance dried by quicklime is approximately that of
a butyleneglycol, but the percentage of carbon is about 0°6 too low.
It does not reduce Fehling’s solution either in the cold or on heating.
That this substance contains a large proportion of 2:3-butyleneglycol,
CH;.CH(OH).CH(OH).CHs, is shown by the following facts :—
1. When the liquid is heated with phenylisocyanate dissolved in anhydrous
ether, combination occurs and a mixture of urethanes is produced. The
fraction of these which is least soluble in aleohol comprises about 90 per cent.
262
402 Dr. Harden and Mr. Walpole. Chemical Action of [Dee. 5,
of the whole amount and has the composition of the diphenylurethane of
butyleneglycol (CsHy)02.2CsH;NCO) :-—
Analysis.
Found. Calculated.
CEO Res: | 65°79 65°85
Le anatase ae 6°21 6°09
Nets ont 8:57 8°35
It is sparingly soluble in cold alcohol, ether and benzene, crystallises in
rosettes of needles and melts at 197° to 198° (uncorr.). When the urethane
is boiled with baryta water or caustic soda solution it is decomposed and
yields a glycol boiling at about the same temperature as the original material.
This glycol has, however, not yet been isolated in the perfectly pure and dry
state. A monourethane, CsH,)02CsH;NCO, has also been prepared which is
somewhat more soluble in cold alcohol than the diurethane, and crystallises.
in needles, melting at 100°:—
Analysis.
Found. Calculated.
IN GS ae eeeeene 6°89 6°65
2. Both the crude glycol and that recovered from the diurethane are con-
verted by oxidation with bromine in the light* into diacetyl, CH;.CO.CO.CH;.
which was recognised by its extremely characteristic appearance and smell,
and by the formation of a phenylosazone melting at 242° to 242°8 (uncorr.).+
The formation of this substance shows conclusively that 2 : 3-butyleneglycol
must be present in the fermentation product.
Detection of Acetyliethylearbinel among the Ferinentation Products—1t was
further found that the distillate from the liquid in which the organism was
grown reduced Fehling’s solution in the cold and gave with phenylhydrazine
the osazone of diacetyl, melting at 243°. These properties point to the
presence in the distillate of acetylmethylearbinol, CH3.CO.CH(OH).CHs,
which has previously been detected in this way by Grimbert? and by
Desmots§ in the products of the fermentation of glucose by several bacteria =
B. tartricus, B. mesentericus vulgatus, B. fuscus, B. flavus, B. niger,
B. ruber, B. subtilis, and Tyrothrix tenuis. It has also been found in
vinegar.
This compound appears only to be formed in very small amount. Since it
* y. Pechmann, ‘ Ber.,’ 1890, vol. 23, p. 2427.
v. Pechmann, ‘ Ber.,’ 1888, vol. 21, p. 2754.
‘Compt. Rend.,’ 1901, vol. 132, p. 706.
‘Compt. Rend.,’ 1904, vol. 138, p. 581.
GK ++
1905. | B. lactis aerogenes on Glucose and Mannitol. 403
is likewise converted into diacetyl by oxidation with bromine in the light,
it is important to notice that the glycol used for conversion into diacetyl], as
described above, was quite free from any substance capable of reducing
Fehling’s solution, and yielded a relatively large amount of diacetyl.
Il. Action of B. lactis aerogenes on Mannitol.
A quantitative examination of the products of fermentation of mannitol by
B. lactis acregenes showed that in this case also the action differed from that
produced by B. coli communis, but that the deficit of carbon was only one-half
of that found for glucose. This is shown in the following tables: Table III
giving the percentages and Table IV the number of carbon atoms per molecule
of mannitol represented by the products in two experiments (Cols. 1 and 2).
As before, the products obtained by the action of B. coli communis are also
given for the sake of comparison (Col. 3) :—
Table IIT.
] 2: 3
PAS COMO Ree: ei ataciriess tse crac nacaeeweess 32°5 32°35 281
eAtceticraciduuns. passe dacissuacnensennss 2°5 2:1 9°5
Malcticlacidhee gt: .: cccosassteeauteens 8°6 8°6 18 6
DEANE EXO! LoonssoncaosasoononEobed 3:2 2°8 8:9
ING ETC EYENGl ao onosossboSocsabeobdac iS 1°6 3°0
Carbon dioxide..........0cecsseeeeeee 3DEo) 35 °5 28 44
Carbon dioxide, c.c. per gramme} 180°3 180 °3 143 -0
Hydrogen, ¢.c. per gramme ...... 138 °3 143 6 167 0
TOs Gy OXO), Soccocooopoponboodocgnen 0°77 0:79 1:18
Percentage excess of 1-lactic acid 65-0 56 0 79-0
J
Table IV.
| 1. | 2 | 3.
ACOH OMA Rie es metaren tect erciens 2°57 Jeo" 2°22
BATCETIC ACL sa vase Suis cae ce canaeneete 0°15 0:12 0°58
Macticvacid! eta. swccnsacaceekeeceecees 0°52 0°52 1°13
SHC OUAG GO!” cooononsoncnnooeooenboCo 0-20 0°17 0°55
HOnmic acids ssaecee see 0:06 0 :065 0:12
Carloon dioxide...............0.c0 eee 147 1°47 1°16
Totally saiscecc | 4:97 491 5°76
| H atoms per molecule glucose al 2-26 | 2°34 | 2°7
| PVs why |
404 Action of B. lactis aerogenes on Glucose and Mannitol. .
Further examination has shown that in this case as in that of glucose, both
acetylmethylearbinol and a glycol are produced, but both in much less
quantity. The amount of crude glycol actually isolated from the products
of fermentation of 50 grammes of mannitol was only 0°75 gramme. Since,
however, the loss in isolating may be roughly taken as about 5 grammes, this
is approximately the yield which would be expected if 6 to 7 grammes were
formed. The nature of these products and their quantitative estimation, as
well as the study of their optical properties, is still under investigation, and
search is also being made for these and similar substances among the fermen-
tation products of other bacteria.
General Considcrations.
The production of so large a proportion of 2:5-butyleneglycol in these
experiments affords clear proof that this substance is derived from the
glucose. The interesting question as to the mode of its production from
the glucose or mannitol molecule will be best deferred until a more complete
examination of the products, and especially of their optical relations, has
been made. The close constitutional relation between the glycol and lactic
acid, and the readiness with which its oxidation product —diacety1—passes
into an aromatic compound are also points of great interest. It may,
however, be noted that the comparison of the fermentation products of
Bb. coli communis and B. lactis aerogenes shows, firstly, that the alcohol
produced by the latter organism is slightly greater in amount than that
due to the former, and, secondly, that it is at the expense of that part
of the molecule which in the B. coli fermentation yields acetic.acid and
lactic acid, that the B. lactis aerogenes forms the new products.
It may further be observed that both these bacteria produce twice as much
alcohol from mannitol as from glucose, a fact which tends to confirm the
suggestion previously made,* that the formation of alcohol in these reactions
is related to the presence of the terminal CH.(OH).CH(OH) group, which
occurs twice in the molecule of mannitol and only once in that of glucose.
A substance of the composition of butyleneglycol has previously been
isolated from the products of fermentation of sugar by yeast,f and was also
found in winef and. in brandy.§ This substance, boiled at 178° to 179°,
yielded a diacetin boiling at 192° to 193°, and was considered to be identical
* Harden, ‘ Trans. Chem. Soc.,’ 1901, p. 601.
+ Claudon and Morin, ‘ Compt. Rend.,’ 1887, vol. 104, p. 1109 ; Henninger and Sanson,
‘Compt. Rend.,’ 1888, vol. 106, p. 208.
{ Henninger, ‘Compt. Rend.,’ 1882, vol. 95, p. 94.
§ Morin, ‘Compt. Rend.,’ 1887, vol. 105, p. 1019.
The Alcoholic Ferment of Yeast-Juice. 405
with the synthetical isobutyleneglycol of Nevolé,* which boils at 176° to 178°.
The yield obtained from sugar was, however, very small, and only amounted
to about 0:2 per cent. after allowing for the losses involved in the extraction
of the compound,
In view of the properties of the crude glycol described above, it would seem
advisable to re-examine Henningevr’s glycol, the constitution of which was not
experimentally examined.
The Alcoholic Ferment of Yeast-Juice.
By Artuur HarpeEn, D.Sc., Ph.D., and WILLIAM JoHn Youne, M.Sc.
(Communicated es Dr. C. J. Martin, F.R.S. Received December 8, 1905,—Read
February 1, 1906.)
(From the Chemical Laboratory, Lister Institute.)
1. Effect of the addition of Boiled and Filtered Yeast-jwice on the Fermentation
of Glucose Produced by Yeast-juice.
In the course of some experiments on the action of various proteids on the
fermentative activity of yeast-juice, it was observed that the alcoholic
fermentation of glucose by yeast-juice is greatly increased by the addition of
yeast-juice which has been boiled and filtered, either when fresh or after
having undergone autolysis, although this boiled liquid is itself incapable of
setting up fermentation. Thus, the total fermentation produced by yeast-
juice acting on excess of glucose is, as a rule, doubled by the addition of an
equal volume of the boiled juice, and a further increase is produced when a
greater volume is added, the sugar concentration being kept constant.f
A similar observation was previously made by Buchner and Rappj in a
single experiment (No, 265).
The following table embodies a few of the results obtained, the yeast-juice
being prepared and the amount of carbon dioxide evolved being estimated by
* “Compt. Rend.,’ 1876, vol. 83, p. 65.
+ Harden and Young, Preliminary Note, ‘Proc. Physiol. Soc.’ 1904, vol. 32,
November 12.
{ ‘ Ber.,’ 1899, vol. 32, p. 2093.
406 Dr. A. Harden and Mr. W. J. Young. [ Dec. 8,
the method previously employed by the authors.* In every case the con-
centration of sugar was kept constant, and both in these and all the
fermentation experiments described in this paper, toluene was added as an
antiseptic. ce
Table I.—Effect of the Addition of Boiled Yeast-juice on the Total
Fermentation of Glucose by Yeast-juice.
| No. | Juice. Water. ae Glucose. Time. eae
C.e. C.c. c.c, grammes. hours. gramme.
1 25 25 (0) 5 72 0-137 |
a oe 25 (0) 25 5 72 0 °378
2 20 20 (0) 4 44 0°115
pas 20 (0) 20 4 44, 0 °363
3 25 0) (0) 2°5 40 0 °370
Eo ae 25 (0) 25 5 40 0-620
d 20 40 0 6 42 0-458 |
aad 20 0) 40 6 42 0 °858
" 25 25 0 5 44, 0 °346
AE 1 25 0) 25 5 44, 0-709
6 25 25 0 5 48 0°110
ee 25 0 25 5 48 0-216 |
7 25 25 0 5 60 0°273 |
Bees 25 (0) 25 5 60 O -466 |
8 f{ 25 25 0) 5 120 0-424
spate | 25 0 25 5 120 0-959
25 25 @) 5 72 0 °414
25 20 5 5 72 0-546
Queers < 25 10 15 5 72 0°735
| 25 5 20 5 72 0°810
25 0 25 5 72 0-924
(25 25 ny) 5 70 0-246
25 O 25 5 70 0 °356
10 3 25 50 0 VES 70 0-180
sores | 25 0 50 7°5 70 0-431
| 25 75 ) 10 70 0-141
| 25 (0) 15 10 70 0°515
In Experiments 1 to 5 the juice added had been autolysed before being
boiled; in Nos. 6 to 8 the added juice was boiled as soon as it had been
prepared. Experiments 9 and 10 show that each successive addition of
boiled juice, from 0-2 to 3 volumes, produces a further increase in the amount
of the fermentation.
A similar effect is produced, (1) By the precipitate produced in boiled yeast-
juice by the addition of 3 volumes of alcohol (Experiment 1, Table II);
(2) By the liquid formed by the autoplasmolysis of yeast, when it is allowed
to stand at the air temp2rature for some time (Experiments 2 and 3, Table IT) ;
(3) By the liquid obtained by boiling Buchner’s “ Aceton-Dauerhefe” with
* Harden and Young, ‘ Ber.,’ 1904, vol. 37, p. 1052.
1905. | The Alcoholic Ferment of Yeast-Juace. 407
water (Experiment 4, Table II). Further, yeast killed by acetone and ether
(Aceton-Dauerhefe) reacts with boiled juice in the same way as does yeast-
juice (Experiment 5, Table IT).
Table II.—Effeet of Various Substances in Increasing Alcoholic
Fermentation.
i a Uae Ween : lee Sa! ss
_ | Yeast- Ke ; ; Carbon
No, ice! Addition. Glucose. Time. | dioxide.
| |
7 ae Ol ; |
; Ci: ; hours. gramme.
1 25 713) CYGn WERE pecobocoooccoccunbocccdEe0 3) 48 0-110
25 | 25 c¢.c. water + precipitate by 5 48 0-268
_ 75 per cent. alcohol from 25 c.c.
. boiled fresh juice
25 Filtrate from 25 e.c. boiled fresh 5 48 O-'141
juice + 3 volumes alcohol, made |
to 25 c.c.
25 25 c.c. water + precipitate from 5 48 0-286
25 e.c. boiled old juice by
75 per cent. alcohol |
i |
2 25 OS GGL IEE pant ocosoban coh ane ebabeccns 5 | 72 0-070
25 25 c.c. autoplasmolysed yeast- 5 72 0-189
juice, made neutral
Ee eae TDG I0.c. water es cco les. tees vessces 5 72 0-084
25 25 ¢.c. autoplasmolysed yeast- 5) 72 0-172
juice, made neutral
DOW A 2otGIG! waters. f.sccankccctas sed: cecedos 5 72 0°475
4 25 | 25 ¢.c. aqueous infusion of 5 72 | 0-625
2 grammes Aceton-Dauerhefe |
5 |2 grammes) 40 c.c. water .........:.ccceeecede eee ens 4 48 | 0:062
Aceton- |
Dauerhefe |
" | 20 c.c. water + 20 c.c. boiled juice | 4. | 48 0-136
2. Dialysis of the Boiled Juice.
The constituent of the boiled and filtered juice to which this effect is due
is removed when the liquid is’ dialysed in a parchment tube, leaving an
inactive residue. In the experiments detailed in the following table
(Table III, Experiments 1, 2 and 3) the effect of the addition of boiled juice
is compared with that produced by the residue and dialysate respectively.
In Experiment 4, the unboiled juice was dialysed, and the fact that the
dialysate had a similar ettect to a boiled juice shows that the active
constituent exists in the original yeast-juice and is not formed during the
boiling.
408 Dr. A. Harden and Mr. W. J. Young. [Dec. 8,
Table III—Dialysis of Boiled Yeast-juice. 25 cc. yeast-juice+5 grammes
glucose + toluene.
| No. | Water. ola Residue. Dialysate. | Dime, Carbon
| juice dioxide.
c.c. C.c. c.c c.c. hours. gramme.
1 25 ) ) i) 48 0-253
@) 25 (0) 0 48 0°561
0 0 25 oO 48 0 :264
2 25 0 (6) 0 4s 0-268
0 25 i) ) 48 0-497
i) ) 25 0 48 0-276
Verse en 25 ft) 0 0 72 0-113
) 25 0 0 72 0 +334.
i) 0 25 ) 72 0-189
c 0 0 25 72 0-334
| 4 25 0 0 0 48 0-154
i) ) i) 25 48 0-251
3. Dialysis of Yeast-juice.
The facts above detailed suggested the possibility of dividing yeast-juice
into two fractions by dialysis; an inactive residue and a dialysate which,
although itself inert, would be capable of rendering this residue active.
This was experimentally realised by filtering the juice through a Martin
gelatin filter.*
This method of rapid dialysis was chosen because the yeast-juices at our
disposal lost their activity too rapidly to permit of the ordinary process of
dialysis through parchment being carried out. Hither a 10- or a 7:5-per-cent.
solution of gelatin was used to impregnate the Chamberland filter and the
filtration was carried out under a pressure of 50 atmospheres.
Only a portion of the juice placed in the filter was actually filtered, the
remainder being simply poured out of the case as soon as a sufficient quantity
of filtrate had passed through. The residue adhering to the candle, which
consisted of a brown viscid mass, was dissolved in water and made up to the
volume of the juice filtered. Glucose was then added and one portion
incubated at 25° with an equal volume of sugar solution and a second portion
with an equal volume of the filtrate or of a boiled juice, containing an equal
amount of glucose. Before incubation the carbon dioxide was pumped out of
all the solutions. The filtrate was invariably found to be quite devoid of
fermenting power, none of the enzyme having passed through the gelatin.
* “Journ. Physiol.,’ 1896, vol. 20, p. 364.
1905. | The Alcoholic Ferment of Yeast-Jwice, 409
The results (fable [V) show that in this way an almost inactive residue can
be obtained which is rendered active by the addition of the filtrate (Experi-
ments 1, 2, 3) or a boiled juice (Experiment 4).
Table [V.—Filtration of Yeast-juice through the Martin Gelatin Filter.
1d.¢.c. residue+3 grammes glucose + toluene.
No. | Water. | Filtrate. | Boiled juice, Time. Carbon dioxide.
|
c.c. ° cc. c.c. hours. | gramme
1 15 0 0 i G45. 0-000
0 15 0 Fee | 0-035
2 15 0 0 60 | 0-001
0 15 0 liner TEOMA | 0-051
3 15 0 0 60r| 0-008
0 15 0 60 | 0-064
4 15 0 0 60 | 0-024
0 0 15 60 | 0-282
The total fermentations observed even in the presence of the filtrate are
very low, this being, at all events in part, due to the fact that in this series
of experiments the original juices themselves happened to be of low
fermenting power.
In a second set of experiments (Table V) a smaller quantity of juice was
placed in the filter and the filtration was continued until no more liquid
would pass through. The residue was then washed several times by adding
water and forcing it through the filter. The time occupied in this process
varied greatly with different juices, the limits for the filtration and washing
of 50 cc. of juice, using two filters simultaneously, were about 6 to 12 hours.
The carbon dioxide was not estimated by absorption in potash as in the
previous cases, but was collected and measured over mercury, by means of
the apparatus described later on, the object of this procedure being to ascertain
not only the total amount of carbon dioxide produced, but the rate and
duration of the evolution. The residue was dissolved in water and made to
the same volume as the originai juice, and the filtrate was evaporated down
to the same volume. All the solutions were saturated with carbon dioxide at
the temperature of the bath (25°) before the measurements were commenced,
and the observations were continued until all fermentation had ceased.
The boiled juice added in Experiments 1, 3 and 4 (Table V) was obtained.
by boiling a portion of the same preparation as was used for the filtration.
The carbon dioxide is expressed in cubic centimetres under atmospheric
conditions.
410 Dr. A. Harden and Mr. W. J. Young. [ Dec. 8,
Vol. of i ue yen Boiled i Carbon
No. juice filtered. Wash water.| Residue. | Filtrate. juice. Glucose. dicde:
c.c. c.c. c.c .c. c.c. | grammes, C.c.
1 75 200 25 0 (0) | 25 10 °4
20 (0) 25 5 396 °3
2 80 ‘260 20 (0) (0) 2 8:3
20 20 (0) A 90 -2
e
3 100 250 25 (0) (0) 2°5 0°4
25 (0) 25 5 268
A 50 200 25 @) (0) 2°53 0:9
25 (0) 25 5 192
The process of filtration does not always produce an inactive residue, as on
several occasions the residue after very thorough washing has been found to
retain a considerable amount of activity. No reason has yet been found for
this and it has not yet been ascertained whether it is due to some peculiarity
in the particular specimen of juice or in the special filter employed.
It is of interest to note that in Experiment 2 (Table V) the residue alone
gave 8°3 ¢.c. of carbon dioxide in 3 hours, the amount evolved in the last hour
being only 0-1 cc. At the close of this period the liquid still contained the
alcoholic enzyme, since on the addition of 20 c.c. of the filtrate, fermentation
recommenced and continued for many hours.
These two sets of experiments (Tables IV and V) show that the fermentation
of glucose by yeast-juice is dependent upon the presence of a dialysable substance
which is not destroyed by heat.
4. Analysis of the Effect of the Addition of Boiled Juice upon the Fermentation
of Glucose by Yeast-jusce.
In order to compare the course of the fermentation in the presence and in
the absence of boiled yeast-juice, experiments were carried out in which the
rate of evolution of carbon dioxide was observed in each case throughout the
whole period of activity of the juice, which, as a rule, in presence of an
excess of sugar, lasts for about 48 to 60 hours.
For this purpose the fermentation was allowed to proceed in a 100 cc.
flask, kept at the constant temperature of 25° by immersion in a thermostat,
and connected with an azotometer, in which the gas was collected over
mercury. The gas in the fermentation flask was maintained at a constant
pressure, as nearly as possible that of the atmosphere, by keeping the mercury
1905. | The Alcoholic Ferment of Yeast-Juice. ALI
in the reservoir at a fixed level, by means of a syphon dipping into a small
beaker. The volume of the gas was read on the azotometer without disturbing
the mercury reservoir and was reduced to atmospheric pressure by means of a
calibration curve. Since yeast-juice readily becomes supersaturated with
carbon dioxide, the contents of the flask were vigorously shaken before each
reading of the volume of gas. Before the observations were commenced the
liquids were brought to the temperature of the thermostat, and were saturated
with carbon dioxide. In all comparative experiments the concentration of
glucose was the same.
When the rates of evolution of carbon dioxide from (1) a solution of glucose
in yeast-juice, and (2) a similar solution to which boiled and filtered yeast-
juice has been added are compared, it is found that two phenomena are
concerned in the production of the increased fermentation in the presence of
boiled yeast-juice.
(a) An initial rapid evolution of carbon dioxide is produced, which soow
diminishes until a rate is attained which remains nearly constant for several
hours and is usually, but not invariably, approximately equal to that given
by an equal volume of the same yeast-juice and glucose to which no addition
has been made.
(v) The fermentation rate diminishes more slowly, so that the fermentation
continues for a longer period. The greater proportion of the total increase
is usually due to this second phenomenon.
The results obtained in a typical experiment of this kind are shown in
Fig. 1. The initial period of the evolution is plotted separately (Curves A’
and B’) on a larger scale.
Curves A and A’ in which the evolution of carbon dioxide is plotted
against time represent the course of a fermentation with 25 c.c. yeast-juice
+25 cc. water +5 grammes glucose +toluene. The rate to begin with is
48 ¢.c. per hour, but rapidly decreases until it becomes equal to 24 c.c. per
hour, at which it remains almost constant for about 5 hours, gradually
decreasing until, after the expiration of about 40 to 45 hours, fermentation
ceases. The total evolution amounted to 369 c.c. under atmospheric
conditions.
Curves B and B’ refer to 25 c.c. of the same yeast-juice +25 c.c. of a boiled
yeast-juice +5 grammes glucose+toluene. The initial rate is much higher,
168 cc. per hour, but this falls gradually in the course of 40 minutes to 30 c.c.
per hour. This rate of 30 cc. per hour falls off much less rapidly than that
in Experiment A, the fermentation continuing for about 80 to 85 hours and
yielding in all 1174 ¢.c. of carbon dioxide. It is important to bear in mind
that these curves represent the gradual disappearance of the fermenting power
412 Dr. A. Harden and Mr. W. J. Young. [ Dec. 8,
ea BE? SS
Pf PD a
pe ep
cle a ea acc SIE)
we A ee
ate asa
Oe Se ee
Atcha
ABER ERRRRRERSEE
0 15 20 70 75 80 8 90 95 100)
ES IN HOURS.
—> CARBON D/OXIDE IN C.C.8.
of the liquid, and not the diminution
of the amount of fermentation with
diminishing concentration of sugar, an
excess of this substance being present
throughout.
A comparison of the two curves shows
very clearly the two factors involved
vane cane in the great increase in Experiment B:
jae tf (1) The initial rapid evolution, and
(2) the prolongation of the fermenta-
QO 10 20 30 40 5060 70 80
TIME IN MINUTES —> tion.
5. The Initial Period of Rapid Evolution of Carbon Dioxide.
This is a very striking phenomenon, and a typical example is illustrated in
fig. 2 in which the curves show the course of the evolution of carbon dioxide
(total volume evolved plotted against time) during two hours in the case of:
A. 25 ce. yeast-juice +75 c.c. water +10 grammes glucose + toluene.
B. 25 ec. yeast-juice +50 c.c. water + 25 cc. boiled ae yeast-juice
+10 grammes glucose + toluene.
C. 25 c.c. yeast-juice +75 cc. boiled autolysed yeast-juice +10 grammes
glucose + toluene.
In B and C the initial rates are almost equal (58 ¢.c. in 10 minutes) and
much greater than in A (14 c.c. in 10 minutes). In B the rate rapidly falls
1905. | The Alcoholic Ferment of Yeast-Juice. 413
off whilst in C it diminishes much more slowly. A similar initial period is
also observable in A, but is not nearly so marked.
260
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|
Pile Pete ere a
pee eeeee as ESE dy
ret
Chota ie
P ae ee
: raped
x baa
a
peor
SAP
CARBON D/OX/DE /N C.C.S.
So)
[an}
0 [O97 20> s05"40 —50'— 60° 70-80 (90° V100) “10.7 120
_ TIME 1N MINUTES —w
The extra quantity of carbon dioxide evolved in this initial period may be
calculated by subtracting the amount corresponding with the constant rate
which is finally attained from the total amount observed. This is done
graphically in fig. 2 by continuing the straight line representing the constant
rate back to the axis of ordinates. The following numbers are thus obtained :
for A, 166; for B, 75°4; for C, 192°9.
The amounts due to the addition of boiled juice are therefore: for 1 volume
in B, 75-4—16°6=58'8 ; for 3 volumes in C, 192°9—16:6=176°3 = 3 x 58'8.
The extra amount of carbon dioxide is, therefore, directly proportional to the
volume of boiled juice added.
AlA Dr. A. Harden and Mr. W. J. Young. [ Dec. 8,
6. Production of the Initial Rapid Evolution of Carbon Dioxide by the
Addition of Phosphates.
As the result of a large number of attempts to isolate the constituent of
boiled juice which brings about the increase in fermentation, it was found
that whenever an increase was produced phosphoric acid in the form of a
soluble phosphate was present. The effect of the addition of soluble
phosphates to yeast-juice was, therefore, examined and it was found that a
well-marked initial rapid evolution of carbon dioxide was thus produced.
Since, moreover, the boiled juices employed invariably contained phosphates,
precipitable by magnesia mixture, there can be no doubt that it is to the
presence of these that this initial phenomenon is due. Quantitative estimations
revealed the somewhat surprising fact that the extra quantity of carbon
dioxide evolved in the initial period when a phosphate or a boiled juice is
added, corresponds with the evolution of one molecular proportion of carbon
dioxide for each atom of phosphorus added in the form of phosphate.
In order to obtain accurate results with solutions of sodium or potassium
phosphate, the fact that these absorb carbon dioxide must be taken into
consideration. Solutions of the dihydrogen salts of potassium and sodium are
too acid to be employed and the monohydrogen salts or a mixture of these
with the dihydrogen salts were always used. In every case the liquid before
being added to the yeast-juice was saturated with carbon dioxide at the
temperature of the bath, and the volume of carbon dioxide liberated by the
addition of excess of hydrochloric acid was ascertained in an aliquot portion.
At the close of the fermentation the fermented liquid was acidified and the
residual combined carbon dioxide measured, the difference between this and
the original amount being subtracted from the amount evolved during the
fermentation.
The results are more precise when the yeast-juice employed is an active one,
since when the fermenting power of the juice is low the initial period becomes.
unduly prolonged and the calculation of the extra amount of carbon dioxide is.
rendered uncertain. The equivalence of the carbon dioxide and phosphate is
established by the results contained in the following Table VI. Column i
gives the observed amount of extra carbon dioxide calculated as described
above and reduced to grammes, and Column 2 the equivalent of the phosphate:
added, this being estimated by precipitation with magnesium citrate mixture
in the boiled juice or phosphate solution.
In Experiments 1 to 7 boiled juice was added; in 8 to 14 a solution of
sodium or potassium phosphate.
The maximum rate attained during the initial period is from five to eight
LS (OG ia The Alcoholic Ferment of Yeast-Juice. 415
times as high as the constant rate attained after the evolution of the carbon
dioxide equivalent to the phosphate present.
Table VI.—Equivalence of Extra Carbon Dioxide Evolved during the
Initial Period, and Phosphate added.
Grammes of carbon dioxide. | Grammes of carbon dioxide:
15;-7 560 | ganna ee] eg aD oc 3 om =
ments. Column II— || ments. ap olumn II—
counts 1. Calculated | Comm | Caleulated
* | from phosphate. | ‘| from phosphate.
1 0 090 0 086 Suiid, sOrleG | 0-197
2 0:054: 0 055 9 | 0 066 0 :065
3 0 ‘058 0°051 | 10 | 0-057 | 0-061
4 0 ‘060 0 049 11 0 056 | 0-061
5 0-106 0-112 12 } 0 ‘059 | 0 ‘061
6 0.°103 0-101 | 13 0 ‘068 0-070
7 01138 0-112 | 14 0-071 | 0-070
At the commencement of the period when sodium or potassium phosphate
solution has been added, the rate only gradually acquires its maximum value
and sometimes it only attains this maximum after a considerable interval. —
This phenomenon is occasionally observed in the fermentation produced by
yeast-juice without the addition of phosphate, aud also sometimes occurs, but
to a much smaller extent, when boiled juice is added. It is well shown in
Curve B, fig. 5, which represents the fermentation produced by 25 c.c. yeast-
juice +25 cc. of a 0:06 molar solution of sodium phosphate +5 grammes
glucose + toluene. The cause of this period of induction has not yet been
ascertained.
7. Linit of the Action of Phosphate.
If the fermentation in presence of phosphate be allowed to continue until
the steady rate is attained and a second quantity of phosphate be then added,
a second period of rapid evolution of carbon dioxide sets in and proceeds in a
similar manner to the first. This is shown in Curves B and C, fig. 3, which repre-
sent the effect of the successive addition of two quantities of 5 c.c. of 0:3 molar
sodium phosphate to 25 c.c. yeast-juice +20 c.c. water, in presence of 10 per
cent. glucose. Curve A represents the fermentation in absence of added
phosphate. The phosphate solution employed was a mixture of five
molecules of NaH:PO, with one molecule of NasHPO; and no correction
for combined carbon dioxide was required. The extra amount of carbon
dioxide evolved after each addition is the same, and is equivalent, as
VOL. LXXVIIL—B. Er
416 Dr. A. Harden and Mr. W. J. Young. [Dec. 8,
already stated, to the phosphate added. The equality is shown graphically
in the curve and the equivalence in Experiments 13 and 14, Table VI.
CARBON O/OXIDE /N C.0.8. ——>
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
7/ME 1N MINUTES —> '
This process cannot, however, be repeated indefinitely, as after a certain
limit is reached the reaction no longer occurs and with a large excess the
fermentation is stopped. The exact limit appears to vary both with the
nature of the phosphate added and with the particular specimen of yeast-
juice employed. The greatest amount of carbon dioxide hitherto obtained in
this way from 25 c.c. of yeast-juice is about 0-45 gramme (230 c.c.), which
was observed on two occasions, once after the addition of four volumes of
boiled juice, and again after the addition of 50 c.c. of a solution of a mixed
magnesium potassium phosphate yielding with magnesia mixture 1:187
grammes of magnesium pyrophosphate.
When a specimen of yeast-juice has been incubated until it will no longer
ferment sugar, it is not affected by the addition of phosphate.
The fact that the extra carbon dioxide calculated in this way is equivalent
to the phosphate present, suggests the superposition of two actions.
Whether this is to be explained by the presence of two distinct enzymes or
simply by the increased activity of a single enzyme remains to be decided.
1905. | The Alcoholic Ferment of Yeast-Juce. 417
8. Products of Fermentation in the Presence of Phosphate.
The carbon dioxide evolved during the initial period after the addition of a
phosphate is the product of a true alcoholic fermentation of the glucose, in
which alcohol and carbon dioxide are produced in equivalent amounts. This
was proved in the following way. Twenty-five cubic centimetres of a solution
containing 25 grammes of glucose and 5 c.c. of a 0°3 molar solution of
potassium phosphate were added to 25 c.c. of yeast-juice; the mixture was
incubated and the carbon dioxide collected and measured.
As soon as the rate of evolution had become constant, a further addition
of 10 cc. of 0°3 molar phosphate solution was made and the fermentation
again continued until the rate had become constant. The gas evolved was
tested and found to be carbon dioxide. The total amount evolved during the
experiment, which lasted for 2 hours 10 minutes, was 163-4 cc. at 19°-6 and
758°6 mm. or 0:291 gramme, the equivalent of the phosphate added being
0-196 gramme. The liquid was then distilled with steam and the alcohol
estimated in the distillate, 1312 grammes being found to be present.
Twenty-five cubic centimetres of the original juice were found to contain
0:983 gramme of alcohol and therefore 1°312—0:983 = 0°329 gramme were
formed by the fermentation of the sugar. The ratio of alcohol to carbon
dioxide produced is therefore 0°329/0:291 = 1:13, which agrees well with the
ratio previously found by similar methods for the fermentation of glucose by
yeast-juice.* The theoretical ratio is 1:04.
Lactic acid and acetic acid were also estimated in the original juice and
after fermentation in presence of phosphate, but only a very small variation
was observed. Twenty-five cubic centimetres of juice gave before fer-
mentation 0122 gramme of zinc lactate and 0:083 gramme of acetic acid,
and after fermentation 0°102 gramme of zinc lactate and 0:072 gramme
of acetic acid.
9. Fate of the Phosphoric Acid.
When the fermented liquid is boiled and filtered almost the whole of the
phosphorus present is found in the filtrate, but it is nearly all in a form
which is not precipitated by ammoniacal magnesium citrate mixture.
In the following experiment three quantities of 25 c.c. of yeast-juice were
taken :—
A. Hot water was added, the solution heated in a boiling water-bath and
the coagulate filtered off and well washed.
B. Ten cubic centimetres of a 30 per cent. glucose solution and 10 c.c. of
* Harden and Young, ‘ Ber.,’ 1904, vol. 37, p. 1052.
an elees
418 Dr. A. Harden and Mr. W. J. Young. [Dec. 8,
0:3 molar potassium phosphate solution were added and the liquid at once
heated to the boiling point, filtered, and the coagulate washed.
C. The same additions were made as to B and the liquid then fermented
until the close of the initial period, after which it was heated and filtered
like the others.
The total phosphorus was then estimated in each of the coagulates and in
each of the filtrates, and the phosphorus precipitated by magnesium citrate
in each of the three filtrates. The estimations of total phosphorus were
made by heating with sulphuric and nitric acids until colourless, diluting and
precipitating with magnesium citrate mixture in presence of excess of
ammonia.
The following were the results obtained, the numbers representing the
grammes of magnesium pyrophosphate per 25 c.c. of juice.
Table VII.
A. B. C.
SOAS etn Juice + phosphate. | Juice + phosphate.
| Original juice.) Not fermented. Fermented.
Coagulate ............0.. 0053 0-057 0-072
Filtrate—
(a) Precipitated by
Meg citrate ......... 0 °126 0-480 0:070
(6) Not precipitated
by Mg citrate ... 0:271 0-282 0-679
Total -..... 0-450 0°819 0 °821
|
The amount of phosphate added was equivalent to 0372 gramme of
magnesium pyrophosphate. ,
A number of other results are given to show the extent to which
phosphate is converted into the non-precipitable form by this reaction.
All the estimations were made by boiling and filtering the fermented liquid
immediately upon the close of the initial period. As before the numbers
represent grammes of magnesium pyrophosphate obtained from 25 c.c. of
juice.
The form in which this non-precipitable phosphorus is actually present in
the fermented liquid, and in the liquid which has been boiled and filtered,
has not yet been ascertained with certainty. Experiments which are still in
progress, however, appear to indicate that it exists in combination with
glucose, probably in the form of a phosphoric ester. ‘
1905. | The Alcoholic Ferment of Yeast-Juice. 419
Table VIII.—Conversion of Phosphate into the Non-precipitable Form by
Yeast-juice and Glucose.
Phosphate Precipitable Non-precipitable
added. phosphate in filtrate. | phosphate in filtrate.
1 0553 0 ‘066 1 °032
2 0-490 0-090 0 832
3 0-250 0054 0 °685
4 0-488 0-091 1-040
5 0 °495 0-088 0-881
The question as to whether the entire phenomenon of the fermentation of
glucose by yeast-juice depends on the presence of phosphates has not yet
been definitely decided. The addition of phosphate undoubtedly produces
a larger increase ,in the total fermentation than is simply due to the
equivalent amount of carbon dioxide evolved in the initial period. The
extent of this increase appears to vary very considerably with different
specimens of yeast-juice, but the prolongation of the fermentation is not so
great as is caused by boiled fresh juice. This question can only be
satisfactorily settled by ascertaining whether the addition of a phosphate to
the perfectly inactive residue obtained from a juice by filtration through a
gelatin filter is sufficient to restore its fermenting power in the same way as
the filtrate or a boiled juice. Experiments on this point are in progress, but
no decisive result has as yet) been obtained, and all discussion of this point
will best be deferred until these are completed.
Various other points of interest raised in the course of the investigation,
and the study of the relation of these phenomena to the fermentation of
glucose by living yeast, are also occupying our attention.
A short outline of the main conclusions arrived at in the foregoing paper,
has been previously published in the form of two preliminary com-
munications, without any experimental details.* After the appearance of
these notes, Buchner and Antoni}+ repeated and contirmed a number of the
experiments dealing with the effect of boiled juice and of phosphates on the
total fermentation, and with the separation of the juice by dialysis into an
inactive residue and a dialysate capable of rendering it active. Buchner
and Antoni were able, with the more stable juice at their disposal, to carry
out the dialysis in the ordinary way for 24 hours and in this manner to
confirm the results obtained by the use of the gelatin filter. Owing to the
* ‘Journ. Physiol.,’ 1904, vol. 32 ; ‘ Proc.,’ of November 12; ‘Proc. Chem. Soc.,’ 1905,
vol. 21, p. 189, June 6.
t ‘Zeit. Physiol. Chem.,’ 1905, vol. 46, p. 136.
420 Mr. Armit and Dr. Harden. Estimation of — [Dee. 5,
lack of experimental detail, Buchner and Antoni imagined that in our
comparative experiments the concentration of glucose and of enzyme had
not been kept constant, and ascribed part of the increase produced by boiled
juice to the favourable effect of a diminution in the concentration of the
sugar and of the alcohol, which is always present, by dilution with the added
boiled juice. The details given above show that neither of these influences
had any share in the effects observed by us.
The Quantitatie Estimation of Small Quantities of Nickel mn
Organic Substances.
By H. W. Armit and A. Harpen, D.Sc., Ph.D.
(Communicated by Dr. C. J. Martin, F.R.S. Received December 5, 1905,—
Read February 1, 1906.)
_ (From the Chemical Department of the Lister Institute of Preventive Medicine.)
In the course of an investigation into the toxic action of certain nickel
compounds, it was found necessary to devise a method of detecting and
estimating nickel, when included in animal tissue, in quantities not exceeding
a few milligrammes per cent.
A method has therefore been worked out, which, although in many respects
only differing from the usual methods in virtue of slight alterations of
detail, is capable of demonstrating extremely small quantities of nickel
accurately. The method may be divided into three stages: (1) The Ashing ;
(2) The Separating ; and (3) The Estimating stages.
1. Ashing.—The substance to be examined must be placed in a porcelain
crucible (platinum is unsuitable, as a considerable loss of nickel takes place,
probably by an alloy of platinum and nickel being formed) and evaporated
to dryness over a water bath. If the substance be solid, it should be cut up
into small pieces. The crucible is then heated carefully with a Bunsen flame,
but it may be wise to further dry in a hot air oven or on a sand bath before
this. Then it is burned over a Fletcher burner, and lastly fully incinerated
in the blow-pipe flame. With some care, it is possible, as a rule, to oxidise
fully all the carbon, without recourse to any foreign material. The crucible .
is then placed on the water bath, and 10 ¢.c. of pure hydrochloric acid are
added and allowed to evaporate to dryness, this process being repeated.
The residue is then extracted with water to which a small quantity of hydro-
1905.] Small Quantities of Nickel in Organic Substances, AZL
chloric acid is added. For this purpose, 2 c.c. of a four times normal acid
are generally employed. The extract is then filtered. The ash so obtained
is practically completely soluble.
Ashing by Kjeldahl’s method, or better, with sulphuric and nitric acids,
can also be employed, but has two disadvantages over the simple incineration
method: Firstly, it takes longer; and secondly, it introduces foreign salts,
which should, if possible, be avoided.
2. Separation.—Firstly, it is necessary to get rid of the iron and at the
same time of the phosphates. Those tissues, which contain iron in excess,
eg., blood, may be treated by precipitation with excess of ammonia and
filtration. The process should be repeated three times, the precipitate being
redissolved each time with the same quantity of acid as was used for the
extraction. If some of the iron separates out from the filtrate on being
evaporated, it may be necessary to refilter before dryness is reached. When
the substance contains little or no iron, it is necessary to add a sufficiency
to combine with the quantity of phosphates present. If the phosphates are
present in excess, the following method is employed. The cold solution
is made neutral to litmus or very faintly acid with ammonia. An excess
of ammonium acetate is then added (as a rule 8 to 10 cc. of a 10-per-cent.
solution suffices), and sufficient ferric chloride to colour the supernatant fluid
yellowish-red. The mixture is then boiled, when all the iron separates out
as phosphate and basic acetate. For those tissues yielding large quantities of
phosphates, ¢.g., liver, the amount of ferric chloride necessary is comparatively
large, and some difficulty may be experienced with the filtration. The only
possible help is obtained by using two or more filters. The washing of the
precipitate must be carried out carefully, in spite of the considerable loss of
time. Precipitation by ammonia may be carried out for the three subsequent
repetitions. The precipitate is each time dissolved in the smallest possible
quantity of acid.
After the united filtrates have been evaporated to dryness, the residue is
again dissolved in water, and dilute hydrochloric acid added, about 6 c.c. of
four times normal acid being usually sufficient. Sulphuretted hydrogen
is then passed through the hot solution for at least half an hour, and it is
then allowed to stand for a time, as the sulphides, which form in acid solution
do not always readily separate out. The liquid is then filtered and the precipi-
tate well washed with sulphuretted hydrogen water. The filtrate is again
evaporated to dryness, re-dissolved in a little water on the water bath and then
a solution of pure sodium hydrate is added in successive portions to the hot
liquid, until no more ammonia comes off. Care should be taken to use
as little sodium hydrate solution as possible, as every sample in the market
422 Mr. Armit and Dr. Harden. Estimation of [Deec. 5,
contains small traces of iron. The nickel is thus precipitated in the form of
the hydrate, and this is converted into nickel sesquioxide by the addition of
1 or 2 cc. of bromine to the cold mixture. The nickel oxide is then collected
by filtration, and after having been well washed, is dissolved in hydrochloric
acid, and the solution evaporated to dryness to remove the excess of acid, and
the residue re-dissolved in water with a-faint trace of acid, in order to prevent
the formation of basic salts. The solution is finally made up to a definite
volume.
In the process of separation, no especial difficulties save the management of
the voluminous iron precipitate, are met with as a rule. At times an
insoluble residue is found on the filter paper when the oxide is dissolved.
This is a trace of a sulphide of copper or another metal of this group, which
has escaped precipitation by sulphuretted hydrogen in acid solution.
5. Estimation—The usual method of quantitative estimation of nickel
colorimetrically is carried out with ammonium sulphide, but it has been found
that sharper results can be obtained by employing a-dimethylglyoxime
CH:;.C(N.OH).C(N.OH).CHs, which was recently shown by Tschugaeff to form
a scarlet red compound with nickel in the presence of ammonia.*
For this purpose, a saturated solution of the reagent in absolute alcohol is
prepared, this is diluted with water until a little of the compound separates
out, and alcohol is then added until complete solution takes place. The fluid
to be tested and a standard solution of nickel sulphate are placed in burettes.
A measured quantity of the fluid is then run into a Nessler tube and to this
0°5 cc. of a 10-per-cent. solution of ammonia and the same quantity
of the dimethylglyoxime solution are added and the whole made up to
30 cc. It is better first to add the ammonia to the nickel solution, then the
dimethylglyoxime, and then allow the colour to develop before diluting up to
the 30 cc. mark. All the solutions must be cold. The fluid becomes
coloured pinkish red, the depth of the coloration depending on the quantity
of nickel present. The most convenient quantity to work with is about
0:08 to 0°01 milligramme. The colour is then compared with that produced
by varying quantities of nickel from the standard solution. The determination
is not complete until a quantity has been found, which gives a colour which is
just too pink, and a second quantity the colour of which is just appreciably
less pink, than the fluid to be tested. The quantity of nickel contained is
then calculated as the amount midway between the two tubes. With a little
practice, it is quite easy quickly to determine very small differences of colour.
The estimation should be concluded as rapidly as is compatible with accuracy,
* ‘Dent. Chem. Ges. Ber.,’ 1905, vol. 38, p. 2520.
1905.| Small Quantities of Nickel in Organic Substances. 423
as, after a short time, the nickel compound with dimethylglyoxime separates
out of the coloured solution as a precipitate.
The advantages of this method over the ammonium sulphide method are :
(1) small traces of iron do not interfere with the final colour, nor with the
sharpness of the method; (2) smaller quantities of nickel can be accurately
estimated ; and (3) it is easier to work in a bad light with the pink than with
the brown colorimetric determination.
Dealing with the colorimetric test alone, with solutions of pure nickel
sulphate, the smallest quantity which gives the reaction is 1/1000 milligramme.
To detect such a small amount, the solution must be placed in the Nessler
tube, then the ammonia and the solution of dimethylglyoxime added, when
one can recognise the characteristic pink colour, and, lastly, the fluid is made
up to 30 ec.; on comparing this with distilled water, a faint but distinct
difference is seen. 3/1000 milligramme gives a recognisable pink colour in
30 cc. of fluid. Working with 0:07 milligramme, differences of 1/1000
milligramme can be recognised with a little practice. This represents a
potential error of + or —0°7 per cent.
In test analyses, serum or blood with nickel sulphate added, the experi-
mental error was kept as low as 2 per cent., using about 1 milligramme of
nickel. Fer example, about 30 grammes of blood were placed in a crucible
and 0:9 milligramme of nickel, in the form of the dissolved sulphate, was
added. After ashing, extracting, removing the copper and iron groups, and
precipitating the nickel in the form of the sesquioxide, etc., the final fluid was
made up to 30 cc.; 2 cc. of this fluid were compared with varying quantities
of a solution containing 0:01 milligramme of nickel per cubic centimetre. It
was found that 59 @.c. gave a colour, which was just too pink, and 5°8 c.c., a
colour, which was not pink enough, so that the 2 cc. contained 0:0585
milligramme of nickel, and the whole solution 0°88 milligramme. This
represents a loss of 0:02 in 0°9, or about 2 per cent.
Electrolysis is only to be preferred when large quantities of nickel are to
be measured, while the method described above is intended for the recognition
and measuring of quantities of nickel not exceeding a few milligrammes.
On Voges and Proskauer’s Reaction for Certain Bacteria.
By Arruur HARDEN, D.Sc., Ph.D.
(Communicated by Dr. C. J. Martin, F.R.S. Received December 5, 1905,—
Read February 1, 1906.)
(From the Chemical Laboratory Lister Institute.)
In 1898 Voges and Proskauer* described a new colour reaction which
they had observed in the case of a bacillus, isolated by Voges and grown in
a medium containing sugar. When potash was added and the tube allowed
to stand for 24 hours or longer at room temperature, a beautiful fluorescent
colour, somewhat similar to that of a dilute alcoholic solution of eosin,
formed in the culture fluid, particularly at the open end of the tube exposed
to the air. The reaction was found to be specific to the bacillus in question,
and was not given by any of the other organisms isolated in the course of
the investigation upon which they were engaged, nor by the B. colt
communis, so that it afforded a most valuable means of differentiation for
the inhabitants of the intestine. Durham} and How} have also employed
this reaction for the discrimination of intestinal bacteria, and MacConkey,§
in confirmation of Durham, has found that out of a large number of bacteria
which were tested only three gave the reaction, these being B. lactis aerogenes
(Kscherich), B. capsulatus (Pfeiffer), and B. cloacw (Jordan).
The examination of the products formed by &. lactis aerogenes from
glucosel| has shown that acetylmethylearbinol, CH;.CO.CH(OH).CHs, and
2°3-butyleneglycol, CHs3.CH(OH).CH(OH).CHs, are both present in the
medium in which this has been cultivated in the presence of glucose. The
acetylmethylcarbinol has not as yet been isolated in the pure state, but
is present in the aqueous distillate obtained by distilling the culture medium.
This distillate and the glycol were, therefore, treated with caustic potash in
order to ascertain whether either of them was the cause of the reaction
just described. Neither of these substances produces the characteristic _
fluorescent coloration with potash alone, but when peptone water is also
added, acetylmethylearbinol gives the reaction after standing for about
24 hours, whilst the glycol does not react in this way even on standing.
The coloration was produced in the characteristic manner described by
‘Zeitschr. f. Hyg.,’ 1898, vol. 28, p. 20.
‘Journ. of Experimental Medicine,’ 1900—1901, p. 354.
‘Centralbl. f. Bakter.,’ 1904, vol. 36, p. 484.
‘Journ. of Hyg.,’ 1905, vol. 5, 349,
|| Harden and Walpole.
Hot+ +
On Voges and Proskauer’s Reaction for Certain Bacteria. 425
Voges and Proskauer, commencing at the open end of the tube exposed to
the air. This suggests oxidation as a factor in the phenomenon, and as
acetylmethylearbinol is very readily converted by oxidation into diacetyl,
CH;.CO.CO.CHs, this substance was tested. Diacetyl yields the fluorescent
red coloration with peptone water and caustic potash in a few minutes, and
by its aid a much greater depth of colour can be obtained than that observed
with bacterial cultures.
Voges and Proskauer’s reaction, therefore, appears to be due to
acetylmethylearbinol, which is formed by the action of the bacteria on the
glucose of the medium. In the presence of potash and air this is oxidised
to diacetyl, which then reacts with some constituent of the peptone water.
That diacetyl is the active substance and not p-xyloquinone, CsH202(CHs)»,
which is readily formed from it by the action of alkalis, is shown by the fact
that if the diacetyl be allowed to stand for some time with potash solution,
and peptone water be then added, no reaction occurs.
B. cloace (Jordan), which gives Voges and Proskauer’s reaction, was also
found to yield acetylmethylearbinol, which was recognised by its power of
reducing Fehling’s solution in the cold and of yielding the characteristic
phenylosazone of diacetyl with phenylhydrazine.
Acetylmethylcarbinol has also been observed as a product of the action of
certain other bacteria on glucose. Thus, Grimbert* found that it is produced
by £. tartricus, and Desmotsft that it is also formed by the various bacilli
of the mesentericus group and by B. subtilis and Tyrothrix tenuis, These
bacteria should, therefore, give Voges and Proskauer’s reaction, and, as a
matter of fact, B. mesentericus fuscus, the only one which has so far been
examined, gives the reaction quite characteristically when grown in peptone
water containing 2 per cent. of glucose.
A number of other bacteria are being examined and attempts are also
being made to ascertain what constituent of the peptone water it is that
reacts with the diacetyl.
* ‘Compt. Rend.,’ 1901, vol. 132, p. 706.
+ ‘Compt. Rend.,’ 1904, vol. 138, p. 581.
426
A Further Communication on the Specificity and Action in Vitro
of Gastrotoxin.
By Cuarues Bouton, M.D., Research Scholar of the Grocers’ Company.
(Communicated by Professor Sidney Martin, F.R.S. Received January 25,—
Read February, 1, 1906.)
(From the Pathological Laboratory, University College, London.)
[Puates 16 anp 17.]
In July, 1904, I laid before the Royal Society a preliminary communica-
tion on the production of a gastrotoxic serum. In that communication I
stated that the serum, obtained by the injection of the mucous membrane of
the stomach of the guinea-pig into the rabbit, was not speeific in the true
sense of the word ; and further, that I was unable to demonstrate any effect
of the gastrotoxin upon the gastric glands of the guinea-pig in viro,
although necrosis and ulceration of the mucous membrane of the stomach
were produced by injection of the serum into the living animal.
By means of more extensive experiments and improved methods I have
obtained confirmatory evidence that the action of the serum is not truly
specific, and have also been able to demonstrate that a definite effect upon
the gastric cells is produced in vitro. I have also succeeded in preparing a
gastrotoxic serum by injecting the fresh mucous membrane of human
stomach into the rabbit. The present communication is therefore intended
as a continuation of my former one.
The subject will be discussed under the following headings :—
I. Action ta Vitro—
1. Hemolytic action.
. Action upon the gastric granules (agglutination).
. Action upon the soluble proteids of the cells (precipitation).
4. Action upon the intact gastric cells (lysis).
‘oo NOD
If. SPECIFICITY OF GASTROTOXIN—
1. Power of Different Celis to Render the Serum Inactive.
Experiments 77 vivo.
Experiments 7 vitro.
2. Comparison with Entero- and Hepatotozin and Hemolysin.
Experiments 72 vivo.
Experiments ia vitro.
III. Propuction or HuMAN GaAsTROTOXIN.
ITV. GENERAL CONCLUSIONS.
On the Specificity and Action in Vitro of Gastrotoxin. 427
I. ActTION in Vitro.
In my first communication I stated that the blood serum of the rabbit
injected with stomach cells washed free from blood became more highly
hemolytic for guinea-pig’s red-blood corpuscles than it was previous to
injection. I have recently been able to demonstrate that two distinct
heemolysins are produced during the process of immunisation.
The gastrotoxin has likewise the power of producing marked changes in
the soluble proteids, and also in the protoplasmic granules of the gastric
cells. It further brings about slight though definite changes in the intact
cells themselves.
1. Hemolytic Action.
Method.—In obtaining the guinea-pig’s blood corpuscles to test the
emolytie power of the immunised rabbit’s serum, the blood is whipped and
ccentrifugalised, the serum then pipetted off and the corpuscles washed in
0°86-per-cent. salt solution several times. A 5-per-cent. suspension of the
corpuscles in salt solution is used for testing the serum.
A known quantity, usually 1 c.c., of this suspension of corpuscles is mixed
with diminishing amounts of the serum in a series of test-tubes, the volume
of fluid in each tube being made up to the same amount with salt solution.
The tubes are placed in the incubator for one hour, and then in the ice
chamber till the following morning, when the exact point at which the
corpuscles are completely dissolved can be determined with ease.
As an example, the details of the following experiment which was made to
determine the normal hemolytic power of a rabbit’s blood for guinea-pig’s
corpuscles are given :—
oe Normal rabbit’s Cale colation: After 1 nore sea and ice
suspension). serum. chamber.
C.C. ce: c.c.
1 2°75 0°25 Complete solution.
1 2°5 0-5 a
1 2°25 0°75 *
1 2 1 i |
1 1-75 1-25 Almost complete.
1 1°5 1-5 Incomplete.
1 1°25 1°75 es |
1 1 2 e
1 0°75 2-25 i
1 0-5 25 i
: 0°25 2 ue Fluid above corpuscles uniformly
. Ol 2 tinted.
1 Bede 075 2-25 |
1 ties. 1 O08 2-5 Fluid tinted to diminishing heights
: ~ (0-025 2°75 | above corpuscles.
- 0-01 2
1 coat 0075 ioe No laking:
1 - 0-005 2°53
times. 2
1 0 0025 2°75 7
428 Dr. C. Bolton. On the [Jan. 25,
In the test-tube containing 2 cc. serum there was complete solution
of the corpuscles, and in the test-tube containing 0°01 c.c. serum there was a
trace of hemoglobin diffused in the clear fluid just above the deposited
corpuscles.
From the experiment it was therefore found that 2 e.c. serum of this
rabbit would completely lake 1 c.c. of a 5-per-cent. suspension of guinea-
pig’s corpuscles, and that a dilution of the serum of 1 in 400 was the greatest
which would produce any solution at all.
It is not reliable to estimate the hemolytic power solely by finding the
ereatest dilution in which any solution will occur, or in other words the
vanishing point of hemolysis, because this vanishing point may occur in
higher dilutions in the case of a weaker serum than in the case of a stronger
serum. What is the exact reason for this phenomenon does not appear to
be at all clear. Gay (1) has, however, recently shown that in the case of high
dilutions the activity of the complement may be completely inhibited.
Bashford (2) has suggested that in the higher dilutions hemolysis is interfered
with by agglutination of the red corpuscles.
The hemolytic power of most rabbits is fairly constant, but as they vary
somewhat within small limits, I examine the hemolytic power of each
rabbit’s blood before injection. Care must be taken to use exactly the same
dilutions when the serum is subsequently tested, as the amount of dilution
affects the heemolytic power of the serum.
Hemolysis—A few days after the first injection of guinea-pig’s stomach
cells into the peritoneal cavity of the rabbit, the hemolytic power of the
rabbit’s serum for guinea-pig’s red corpuscles is found to have considerably
increased. In one case before injection 2°25 c.c. serum were necessary to
completely dissolve 1 ¢.c. of a 5-per-cent. suspension of corpuscles; seven
days after the injection 0°75 c.c. serum would dissolve the same amount
of corpuscles.
This first increase of hemolysin is a true increase of the natural hemolysin
of the rabbit, because its action like that of the natural hemolysin is
destroyed by heat, and is not restored on adding normal guinea-pig’s serum.
In other words, guinea-pig’s complement will reactivate neither.
At this early stage a slight amount of laking, varying in extent in different
cases, may be seen on reactivating the heated serum with guinea-pig’s
complement, but this laking has never been to any degree extensive enough
to account for the increase of hemolysis. In the above experiment, 21 days
after the first injection, it was found that 0°d c.c. serum would completely.
dissolve 1 c¢.c. suspension of corpuscles, but on heating 2°5 c.c. immune
serum to 55° C., and complementing with 0°25 c.c. guinea-pig’s serum, only
1906.| Specificity and Action in Vitro of Gastrotoxin. 429
the faintest trace of hemoglobin was seen to be diffused in the fluid
immediately above the deposited corpuscles. After the second injection,
however, it is found that guinea-pig’s serum will reactivate the heated
immune serum to a considerable extent. In the above experiment six days
after the second injection 0°5 c.c. heated immune serum, on being reactivated
by 0:25 cc. guinea-pig’s serum, completely dissolved the test amount of
corpuscles. I have confirmed this experiment several times, and to my
mind it conclusively points to the presence of two distinct immune bodies.
(1) An increase of the normal hemolysin of the rabbit which 7s not comple-
mented by guinea-pig’s serum. (2) A newly-formed and therefore artificial
hemolysin which zs complemented by guinea-pig’s serum.
This has an important bearing upon the hypothesis of the multiplicity of
immune bodies, which is upheld by Ehrlich and Morgenroth (3), but is
denied by other observers, notably Muir and Browning(4), and Gay (5)
working in the Pasteur Institute. Agelutination of the red corpuscles also
occurs.
2. Action upon the Protoplasmic Granules of the Gastric Cells.
Method.—The method which I employ in order to demonstrate this action
is an imitation of that described for hemolysin.
The mucous membrane of a guinea-pig’s stomach is first washed free from
blood by sterilised salt solution, which is made to flow through a canula
introduced into the thoracic aorta, the stream issuing from the inferior vena
cava. It is then scraped off, and the pulp ground up in a glass mortar. An
emulsion is made with salt solution and centrifugalised for five minutes at
alow speed. The supernatant fluid on being pipetted off is found to contain
in suspension innumerable large and small protoplasmic granules. The
granules are separated from the albuminous fluid in which they float by
centrifugalisation at a high speed, and repeatedly washing until the
washings give no precipitate with potassium ferrocyanide and acetic acid.
The final suspension of granules in saline solution, which is to be used,
must be well agitated so as to free all the granules, and before use it must
be slowly centrifugalised to ultimately remove any masses of granules or
pieces of tissue which happen to be present.
A series of test-tubes is prepared, each tube containing 2 c.c. of the gastro-
toxic serum in increasing dilutions. The first tube contains undiluted
serum, and the remainder dilutions from 1 in 5 tol in 320. To the contents
of each tube three to five drops, according to concentration of the suspension
of granules in salt solution, are added.
A control of normal saline is prepared, and also a control in which
430 Dr. C. Bolton. On the | [Jan. 25,
0:5 c.c. of the washings is added to 2 cc. serum, so as to eliminate any
possibility of a precipitate of albumin being thrown down from the fluid, in
which the granules are suspended, by the immune serum.
The tubes are incubated for four or five hours and then examined by the
naked eye and also microscopically.
Agqglutination.—A very fine deposit consisting of agglutinated granules is
seen, sometimes only with a lens, at the bottom of the tubes up to a certain
dilution which varies according to the strength of the serum. The super-
natant fluid contains granules in all stages of agelutination, and if the tubes
are allowed to stand in the ice chamber till the next day, all the agglutinated
granules are found to have settled to the bottom.
The reading of the tubes is taken at the end of four or five hours’ ineuba-
tion, and controlled by that taken on the following day, both macro- and
microscopically. The reason for taking the reading twice is because
bacteria are liable to be found in the fluid at the end of 24 hours, since
the scraped mucous membrane cannot be sterilised by heat.
The saline control shows no such deposit or agglutination, and the control
containing the washings shows no precipitate or deposit. The blood of a
normal rabbit shows no deposit and no agglutination, and can therefore be
used as a control.
The amount of deposit is just sufficient to be accounted for by the sub-
sidence of the granules, and on shaking it up the agglutinated granules pass
into suspension, forming a delicate precipitate. Vigorous shaking disentangles
the granules, and the appearance of the solution becomes the same as it was
when the granules were first added.
¢ is thus evident that the serum possesses an action upon the granules
themselves, and that this action is similar to that of the bacterial
agglutinins. The agglutinin appears in the rabbit’s blood in small amounts
about 14 days after the first injection, and can be quite easily recognised
after a second injection.
Lffects of Heat——After exposure to a temperature of 58° to 60° for half
an hour the serum agelutinates gastric granules in as high a dilution as it
does when unheated. In this property of resisting heat the agglutinin
resembles those of bacterial origin.
3. Action upon the Soluble Proteids of the Gastric Cells.
Method.—The solution of proteid is prepared by grinding up the mucous
membrane of the guinea-pig’s stomach, previously washed free from blood,
as described above, in a glass mortar with normal salt solution.
The emulsion is centrifugalised in order to get rid of most of the solid
1906.| Specificity and Action in Vitro of G'astrotoxin. 431
matter, but it is not possible to completely free such a solution from
protoplasmic granules by cenrtifugalisation alone. The solution is filtered
through a Berkefeld filter. The resulting filtrate, which is a perfectly clear
solution like water, gives a precipitate with heat, ferrocyanide of potassium,
and acetic acid, and other precipitants of proteids.
The experiment is done in the same way as that described for the
agglutination test. The gastrotoxic serum is diluted and placed in a series
of test-tubes, and to the contents of each test-tube 0°5 c.c., or even only
two or three drops of the proteid solution, are added. The tubes are placed
in the incubator for four or five hours. Controls of normal rabbit’s serum
and salt solution are also prepared.
Precipitation.—At the end of one hour’s incubation a fine precipitate has
commenced to form in the solution. This precipitate becomes coarser in
appearance, and at the end of about four hours has settled to the bottom of
the tube in considerable quantity, forming a deposit. Flakes of precipitate
ean still be seen floating about in the otherwise perfectly clear supernatant
fluid. Under the microscope this precipitate is seen to consist of amorphous-
looking masses. If the tubes are placed in the ice-box until the next day
the whole of the precipitate will be found to have settled to the bottom,
leaving the supernatant fluid perfectly clear.
As in the case of the agglutination test, I record the result of the
experiment both at the end of four or five hours, and also on the following
day, although in this case one can exclude the presence of bacteria, since
the tubes have been sterilised and plugged with cotton wool, and the gastric
solution filtered.
The control tube containing normal rabbit's serum, and that containing
salt solution, show no precipitate after incubation. It is thus perfectly clear
that the gastrotoxic serum acts chemically upon the soluble proteids of the
guinea-pig’s gastric cells, producing an insoluble compound. This precipitin
appears in the serum about the same time as the agglutinin.
Effects of Heat—As in the ease of the agglutinin, exposure to a tem-
perature of 58° to 60° C. for half an hour does not in the least diminish
the power of the serum to precipitate the soluble proteid, since precipi-
tation occurs in as high a dilution of the heated serum as it does in the
unheated serum.
It is well known that the effects of heat upon the action of precipitins
varies considerably ; some are easily affected, whilst others are resistant.
Action upon Guinea-Pig’s Blood Serum—The experiment is performed in
exactly the same manner as described above. The guinea-pig’s serum is
‘diluted 10 or more times, and 0°5 c.c. added to 2 c.c. gastrotoxic serum.
VOL, LXXVII.—B. 21
432 Dr. C. Bolton. On the [Jan. 25,
The mixture is incubated, and at the end of four hours it is seen that a
similar precipitate has formed. The gastrotoxic serum may precipitate the
serum in as high a dilution as it does the proteid of the gastric cells.
The gastrotoxin does not therefore act exclusively upon the proteid of the
gastric cells.
+, Action upon the Intact Gastrie Cells.
Method—aA_ guinea-pig’s stomach is washed quite free from blood before
removal from the body, as described above. The superficial portion of the
mucous membrane is gently scraped off with a knife and suspended in a
few cubic centimetres salt solution. The test-tube is now very carefully
shaken up for a few seconds and allowed to stand for about 10 minutes,
at the expiration of which time the contents will have separated into two
portions :—
1. A milky fluid.
2. Small pieces of mucous membrane, which either float on the top of the
fluid or settle to the bottom.
The milky fluid is the portion used. It is pipetted off, and on microscopic
examination is found to contain in suspension free cells, masses of cells, and
fragments of glands, together with broken-up cells, free nuclei, and proto-
plasmic granules.
It is quite easy to separate the cells, because on slow centrifulgalisation
the cells and fragments of glands sink to the bottom, but the granules and
nuclei remain in suspension.
The cells are washed to clear away the granules and soluble proteid. A
suspension of cells, masses of cells, and fragments of glands in salt solution
is thus obtained.
The free oxyntic cells are large oval structures with well-defined granules,
but the central cells, which are of a more delicate structure and contain finer
granules, tend to cling together in masses and are easily broken up if too
vigorous shaking is employed.
In doing an experiment three or four drops of suspension of cells are
placed in 2 c.c. gastrotoxic serum in a test-tube, and the mixture incubated
for four or five hours. Controls are prepared with normal rabbit’s serum and
also salt solution.
Lysis—The deposit which has formed in the tube is examined micro-
scopically. I have done a large number of these experiments, and always
with the same results. I have never observed either solution or
agglutination of the cells, such as has been described in the case of other
cytotoxins; in fact, the cells in the serum are much more ‘separated than
1906.| Specificity and Action in Vitro of Gastrotoxin. 433
those in the salt solution, as the latter tend to stick together by reason of the
mucus, which it is impossible to clear away completely.
The cells in the salt solution are quite normal in appearance, but the cells
which have been exposed to the action of the gastrotoxic serum have
become more or less hyaline.
The oxyntic cells are not so much affected as the central cells, the masses
of which appear like pieces of floating glass. The granules are obscured,
and many of the cells look like shadows. The nuclei can usually be seen
except when the cells are massed together.
The cells are examined in the fresh state, as this seems to me to be the
best for practical purposes. I have also stained them with watery solutions
of methyl green, picrocarmine, and safranin, but this does not materially
assist. The stained cells which are affected look like pieces of coloured
glass.
This effect of the gastrotoxic serum appears later than those described
above, and is not observed until after the expiration of about five weeks
after the first injection, four or five injections having been given in the
meanwhile. The effect begins to pass off between three and four months
after the first injection, the animal receiving injections at regular intervals.
The serum of a normal rabbit differs in no respect in its behaviour towards
the gastric cells trom salt solution.
Effects of Heat—A temperature of 55° C. maintained for half an hour
does not destroy the action of the serum upon the gastric cells. It may be
weakened, but only to a slight degree. This result points to the conclusion
that a complement exists in the cells themselves, if the action is due toa
cytolysin of the same construction as a hemolysin. Such endocellular
complements have of course been described before.
I have heated the stomach to 54° C. for half an hour before obtaining the
cells, and have found that in this case the serum produces no effect upon
them. This experiment of course proves nothing, because it is probable that
the vitality of the cells is destroyed by exposure to this temperature, since
cell globulin coagulates at 48° to 50° C.
Removal of Gastrolytic Factor—By saturating the serum with washed
gastric cells of the guinea-pig and allowing the mixture to stand for one hour,
the gastrolytic factor is removed and the action of the serum upon the cells is
destroyed. No change is visible in the cells which have been used to saturate
the serum in the space of one hour, and, since they remove the gastrolysin, it
is evident that the latter becomes anchored to the gastric cells preparatory
to acting upon them. This brings the gastrolysin into line with a hemolysin
in this respect.
212
434 Dr. C. Bolton. On the [Jan. 25,
Il. SPECIFICITY OF GASTROTOXIN.
If this serum be strictly specific for the stomach cells of the guinea-pig, it
should possess chemical affinities for the stomach cells alone and for no other
tissues of the body. On injection into the guinea-pig it would then produce
lesions limited to the stomach, and would also be rendered inactive by
mixture with stomach cells, which would combine with the poisonous
substance in the serum and take it out of solution. Further, other tissues of
the body should not possess chemical affinities corresponding to those of the
stomach cells and, therefore, would not render the serum inactive by mixture
with it, and sera formed against those tissues would not produce lesions in
the stomach on injection.
The specificity has therefore been tested by comparing the relative power of
different cells of the body of the guinea-pig to render the serum inactive, and
also by comparing the effects of the serum with those of a hepatotoxic, an
enterotoxic, and a hemolytic serum obtained by injecting blood.
The experiments have been conducted 2 vivo and in vitro.
1. Power of Different Cells to Render the Serum Inactive.
In my former communication I gave the results of a few experiments. I
have since examined this question more extensively and although the subject
is far from being completed yet I will here give the results of my further
investigations.
In mixing the various cells with the serum to be examined, care must be
taken that enough cells are present to saturate the serum otherwise when
they settle to the bottom of the tube a portion of the serum is left unexposed
to their action. The cells obtained by scraping the mucous membrane of one
guinea-pig’s stomach are enough to saturate 4 c.c. serum, but the resulting
mixture will not yield 4 c.c. serum back. For a reliable experiment it is
necessary to inject at least 10 c.c. serum and, therefore, to obtain this I take
12 cc. serum and mix with it the mucous membrane of three guinea-pigs’
stomachs. After centrifugalisation it is quite easy to obtain 10 c.c. of treated
serum.
The cells of the mucous membrane of the small intestine are obtained in
the same way as those of the stomach.
The liver is pounded up and passed through a tea strainer in order to
prepare it. In all cases the blood is washed out of the organ in question
before it is removed from the body.
If the serum is examined 7m vivo after such treatment a control animal is,
1906.| Specaficity and Action in Vitro of Gastrotoxn. 435
in each case, injected with the untreated serum; if it is examined in vitro
controls are likewise prepared in all cases.
All the experiments have invariably been carried through to a finish on the
same day, and all the tissues and sera used were always obtained fresh on
that day. In testing the hemolytic power of the treated serum equal
weights of the various organs were previously mixed with equal volumes of
the serum. ‘The cells were allowed to stand in contact with the serum for
one hour at laboratory temperature, except in the case of some of the in vitro
experiments, when the tubes were placed in the incubator.
Experiments in Vivo. Mixture with Stomach Cells.—Four experiments have
been done, and in each case with the same result. The gastrotoxin was in all the
cases completely removed by the stomach cells, and the resulting serum pro-
duced no lesions in the stomach at all. The control animals in all cases
showed the usual lesions (see Plate 16, fig. 1).
Mixture with Intestine Cells—In four cases the intestine cells failed to
destroy the action of the serum, the lesions produced by the treated
serum being as extensive as those produced by the untreated serum (see
Plate 16, fig. 2).
In three cases the action was destroyed, but the lesions in the three control
animals were so slight that the toxicity of the serum must have been very
low.
Mixture with Liver Cells—In four cases the liver cells failed to destroy
the action of the serum, but in each case there was a weakening of the
power of the gastrotoxin, judging by a comparison with the effects produced
in the control animals (see Plate 16, fig. 3).
In one case complete removal of the gastrotoxin resulted, and in this case
the latter was of low toxic value, judging by the lesions produced in the
control animal (see Plate 16, fig. 4),
Mixture with Red Blood Corpuscles—In three experiments the action of the
gastrotoxin was unaffected, and in a fourth the lesion was less extensive than
that in the control animal (see Plate 17, fig. 5).
The results of these experiments clearly demonstrate that other organs of
the body besides the stomach have tissue affinities for, at all events, some of
the constituents of this serum, and that they can, if not invariably destroy
its action, at any rate weaken it. On the other hand, the stomach is the only
organ of the body which can invariably and with uniform certainty destroy
the action of the gastrotoxin. The serum is thus not, strictly speaking,
specific, although lesions are not produced in other organs than the stomach
by it as a rule. It may be, however, that one of the constituents of this
complex serum is specific for the stomach to a great extent.
436 Dr. C. Bolton. On the [Jan. 25,
These experiments also illustrate the importance of an organ’s ability to
take up a poison and render it inactive without being itself affected by it.
They likewise explain why large doses of the serum are necessary to produce
the stomach lesions, since a large part of this serum must be rendered inactive
by different organs of the body.
Experiments in Vitro. Hemolysin. Mixture with Stomach Cells of Guinea-
pig.—After treatment with stomach cells the serum shows as high a degree of
hemolytic power as it did before such treatment. It may be higher. At
first sight this result appears to be remarkable, namely, that a cell will not
remove a side chain that is thrown off in response to its injection. In other
words, that side chains may be thrown off which have no affinity at all for
the cells against which they are thrown off.
I have obtained further evidence of the same principle in the case of the
gastrotoxin formed against guinea-pig’s stomach cells by injection of the
rabbit’s stomach cells into the rabbit. The rabbit’s stomach cells will not
remove this gastrotoxin from the serum, and therefore, whether one supposes
that the cytophilic affinity of the amboceptor for the rabbit’s stomach cell is
or is not saturated by an anti-immune body, the fact remains that the rabbit’s
stomach cell has no affinity for the side chain which is active against the
guinea-pig’s cell, and which has been thrown off in response to injection of
rabbit’s stomach cell. Similarly rabbit’s stomach cells will not remove the
gastrotoxin from guinea-pig-rabbit gastrotoxic serum (see Plate 17, fig. 6).
It seems to me most likely that when a cell is absorbed side chains having an
especial affinity for that cell, and which are used in destroying it, are set free
and that other side chains having less affinity for it are set free in diminished
amount, and also side chains having no affinity whatever for it are set free in
smallest amount. In other words, the absorbing cell throws off most of the
varieties of side chains or chemical affinities of which it is possessed, the
number of each being directly determined by the amount of stimulation given
to the particular chemical affinity involved.
The fact that the stomach cells will not absorb the hemolysin is important
from another point of view. I shall show later that the action of a hemolysin,
whatever its origin, is directed especially against the stomach, and also that
lesions due to its action may be limited to the stomach. Now if other organs
of the body have the power of destroying the hemolysin without themselves
being affected, whilst the stomach cells will not absorb it, the result naturally
follows that the hemolysin will be free to act as it may in the capillaries of
the stomach, and therefore will produce lesions.
Mixture with Liver Cells, Intestine Cells, and Red-Blood Corpuscles——Kach of
these three varieties of cells has the power of removing the hemolysin from
1906.| Specificity and Action in Vitro of Gastrotoxin. 437
the serum, and therefore of rendering it incapable of dissolving the red-blood
corpuscles of the guinea-pig in vitro. Occasionally, especially in the case of
the liver, a slight amount of diffused hemoglobin may be seen above the
settled corpuscles.
These experiments clearly indicate that although the hemolytic factor of the
gastrotoxic serum may be of great importance in assisting to produce the
stomach lesions in vivo, yet it 1s not the only one. The reasons for this state-
ment are, that previous mixture with stomach cells will deprive the serum of
its action i vivo, but will not prevent its laking blood corpuscles in vitro ;
that previous mixture with liver cells, intestine cells, or red-blood corpuscles,
although it deprives the serum of its power to lake blood corpuscles in vitro,
will not with any degree of uniform certainty completely prevent its action
in vwo.
Lysin.—I have not yet attempted to compare the gastrolytic strengths of
two sera by determining the highest dilutions in which any action is apparent,
and therefore cannot say whether or not the action in any given case is
diminished. So far as my results go, however, they appear to indicate that,
after exposure of the serum to gastric cells, its action upon such cells is
destroyed, but that after exposure to liver and intestine cells and blood
corpuscles the serum still produces changes in gastric cells in vitro.
These in vitro experiments, so far as they go, point to the same conclusion
as the in vivo experiments—namely, that this gastric cytotoxin is not truly
specific, although one or more of the bodies contained in it may be so, and
that the protoplasmic poisons constituting it have a greater affinity for gastric
cells than for the cells of other organs of the body.
2. Comparison with Hepato- and Enterotoxie Sera and Hemolysin.
The hepatotoxin and enterotoxin were respectively prepared by injecting
the rabbit with the washed and prepared cells of the liver and intestine of
the guinea-pig. The hemolysin was obtained by injecting red-blood
corpuscles,
Experiments in Vivo.—Each of these sera produces hemolytic lesions in the
stomach, leading to destruction of the mucous membrane, the microscopic
condition very closely simulating that due to gastrotoxin (see Plate 17, fig. 7).
They are more uncertain in their action upon the stomach, however, and
this action is liable to be not so strictly limited to the stomach as that of
gastrotoxin. The action of hemolytic serum was described in my previous
communication.
Experiments in Vitro. Hemolytic Power.—Both hepato- and enterotoxin
438 Dr. C. Bolton. On the [Jan. 25,
possess the power of dissolving red-blood corpuscles to about the same degree
as gastrotoxin. They also agglutinate these cells.
Agglutination and Precipitation—The gastrotoxic serum produces similar
effects upon emulsions of liver and intestine granules to those described in
the case of emulsions of stomach granules.
Hepato- and enterotoxin not only act upon emulsions of liver and intestine
granules, but they also act upon emulsions of stomach granules. Hemolysin
obtained by injecting blood has no more power of acting upon these
emulsions than normal rabbit’s blood has.
Lysin—Up to the present no definite hyaline transformation of gastric
cells has been demonstrated as the result of the action of hepato- or
enterotoxin or hemolysin, and gastrotoxin does not appear to act upon the
intact liver or intestine cells.
Removal of the Immune Body by Different Cells——Only one or two experi-
ments of this nature have been done up to the present. In the case of
a rabbit which had been immunised against the red-blood corpuscles of the
guinea-pig, mixture with stomach cells entirely failed to remove the
hemolysin and stomach lesions resulted on the injection of the serum (see
Plate 17, fig. 8). Mixture with liver and intestine cells, however, rendered
the same serum less powerful than before.
In the case of an enterotoxic rabbit, mixture of the serum with either
stomach cells or intestine cells effected complete removal of the amboceptor.
In vitro, stomach cells completely fail to remove the hemolytic factor
from enterotoxic or hepatotoxic serum, as was previously observed in the case
of gastrotoxic serum. liver and intestine cells remove the hemolytic
factor from entero- and hepatotoxin as they do in the case of the gastrotoxin.
The few experiments that have been made with hepato- and entero-
toxin therefore confirm the view that gastrotoxin is not strictly specific.
III. Propuction oF HUMAN GASTROTOXIN.
Nine rabbits have been immunised against fresh human stomach mucous
membrane. Four died from septic infection, the remaining five gave
positive results.
This is not quite such an easy matter as in the case of the guinea-pig’s
stomach, because the supply of human stomach is not constant, and three
weeks or a month may elapse without an opportunity for obtaining the
mucous membrane offering itself. In addition to this the stomach cannot
be used immediately after death, although on one or two occasions I have
been fortunate enough to obtain some from operation cases, and it is
impossible to obtain the stomach free from blood.
1906.| Specificity and Action in Vitro of Gastrotoxin. 439
The normal serum of the rabbit is slightly hemolytic for human blood
corpuscles, but does not produce any effect upon emulsions of human
stomach granules,
I have succeeded in showing that the sera of five rabbits so immunised
became highly hemolytic for human blood corpuscles, and in the one case
in which I tried it, solution of the corpuscles of the monkey also occurred.
The sera also agglutinated and precipitated emulsions of human gastric
granules, and in one case those of the monkey also. Whether hyaline changes
are produced in the cells I have not yet determined.
TV. GENERAL CONCLUSIONS.
The gastric cytotoxin formed in the blood of an animal in response to the
injection of gastric cells thus appears to be a complex body. After a single
injection there is a great increase in the hemolysin normally occurring in
the animal’s blood, and at the same time there is found a new hemolytic
immune body which is not normally present in the animal. The latter is
present in considerable amount after the second injection. The gastrotoxin
also agglutinates red-blood corpuscles. Closely associated with the appear-
ance of this artificial hemolytic immune body is that of an agglutinin which
acts upon the gastric granules, and also that of a precipitin which acts upon
the soluble proteids of the gastric cells.
By repeating the injections these substances are found to be present in
the blood for several months. Whether they are one and the same or distinct
bodies I have not yet proved.
After several injections, and not less than about five weeks from the first,
a further substance appears in the blood, which possesses an action upon the
intact gastric cells. In spite of repeated injections, this substance disappears
from the blood in about four months. It is probably of the same nature as
a hemolysin, but this point requires proof.
The hemolytic factor is only active against blood. The actions of the
agglutinin and precipitin are not confined to the constituents of the gastric
cells, but extend to other proteids of the body. Whether there are separate
agglutinins and precipitins for different proteids, or whether the same
substances act upon all proteids, has not been determined; at all events,
if the same bodies are concerned in all cases, their action upon the proteids
of the stomach cells is probably greater than that upon other proteids.
Whether the gastrolysin itself is truly specific remains to be proved.
The few experiments that have been undertaken in the case of the human
stomach indicate that the human gastric cytotoxin is identical in constitution
with that of the lower animals.
A40 Dr. C. Bolton. On the [Jan. 25,
Note-—The term “ Hemolytic lesions” is used in this paper to signify the
hemorrhages which are produced by the injection of a hemolytic serum ; this
does not however imply that such hemorrhages are directly caused by the
factor in the serum which brings about solution of the red blood corpuscles.
REFERENCES.
1. Gay, “Observations on the Single Nature of H:emolytic Immune Bodies, and on the
Existence of so-called ‘Complementoids,’” ‘Centralbl. f. Bakt., etc,’ 1 Abt.,
Originale, vol. 39, heft 2, 1905, p. 172.
Bashford, ‘ Journal of Hygiene,’ vol. 4, 1904, No. 1, p. 40.
Ehrlich and Morgenroth, ‘ Berl. Klin. Wehschr.,’ 1901, p. 569—598, and 1900, p. 681.
Muir and Browning, ‘ Roy. Soc. Proc.,’ vol. 74, 1904, p. 298.
Gay, loc. cit.
or > go p90
DESCRIPTION OF PLATES.
PuateE 16.
Fic. 1.—Illustrates the removal of the gastrotoxin from the serum by treatment with
guinea-pig’s stomach cells previous to its injection.
Upper Stomach.—From a guinea-pig injected with gastrotoxic serum. Necrosis
of the mucous membrane has therefore resulted.
Lower Stomach.—From a guinea-pig injected with the same dose of the same
serum previously treated with stomach cells. No lesion has resulted ; the
stomach cells have removed the gastrotoxin from the serum by combining
with it.
Fie. 2.—Stomach of a guinea-pig which was injected with gastrotoxic serum previously
treated with guinea-pig’s small intestine cells. The gastrotoxin has not been
removed from the serum by the cells, and the stomach therefore shows
necrosis of the mucous membrane.
Fic. 3.—Stomach of a guinea-pig which was injected with gastrotoxic serum previously
treated with guinea-pig’s liver cells. The gastrotoxin has not been removed
from the serum by the liver cells, and the stomach therefore shows a patch
of necrosis of the mucous membrane. The action of the serum has, however,
been weakened.
Fie. 4.—The lower stomach is that of a guinea-pig which was injected with gastrotoxic
serum previously treated with guinea-pig’s liver cells. The gastrotoxin has
been removed in this case by the liver cells, and the stomach therefore
shows no lesion.
The upper stomach is from the control animal, which was injected with untreated
serum, and shows lesions which are relatively slight.
PLATE 17.
Fic. 5.—Illustrates the fact that guinea-pig’s red blood corpuscles will not remove the
gastrotoxin from the serum by combining with it.
Bolton. Roy. Soc. Proc., B. vol. 77, Plate 16.
[Photographs by B, S, Worrall.]
Bolton. Roy. Soc. Proc., B. vol. 77, Plate 17.
[Photographs by E. S. Worrall.)
1906.| Specificity and Action in Vitro of Gastrotoain. 44]
Upper Stomach.—From a guinea-pig which was injected with gastrotoxic
serum previously treated with blood corpuscles. Necrosis of the mucous
membrane has resulted, because red blood corpuscles will not combine with
the gastrotoxin.
Lower Stomach.—From the control animal, which was injected with the same
dose of untreated gastrotoxic serum, necrosis of the mucous membrane has
therefore resulted.
Fic. 6.—-Ilustrates the fact that rabbit’s stomach cells will not remove the gastrotoxin
from guinea-pig-rabbit gastrotoxic serum.
Upper Stomach.—F rom the control animal, which was injected with untreated
serum, and therefore shows extensive necrosis and hemorrhage.
Lower Stomach—From a guinea-pig which was injected with gastrotoxic
serum, previously treated with rabbit’s stomach cells. The cells have failed *
to remove the gastrotoxin from the solution, and lesions similar to those of
the control animal have resulted.
Fic. 7.—Illustrates the hemolytic lesions which are produced in a guinea-pig’s stomach
by the injection of a guinea-pig-rabbit entrotoxic serum. As seen here, the
lesions are indistinguishable from those produced by gastretoxic serum.
Fic. 8.—Illustrates the fact that guinea-pig’s stomach cells will not remove the
hemolysin from guinea-pig-rabbit hemolytic serum (obtained by injecting
red blood corpuscles). Hemolytic patches are seen in the stomach, which is
that of a guinea-pig. The animal was injected with guinea-pig-rabbit
hemolytic serum, prepared by injecting the red blood corpuscles of the
guinea-pig into the rabbit. Before its injection the serum was mixed with
stomach cells for one hour. The stomach cells have failed to remove the
hemolysin from the serum.
442
The Influence of Increased Barometric Pressure on Man.—No. I.
By Leonarp HI11, F.R.S., and M. GREENWOOD, Jun., M.R.C.S.
Research Scholar of the British Medical Association.
(Received January 16,—Read February 15, 1906.)
Introduction.
The classical researches of Paul Bert,(1) confirmed in recent years by
v. Schrotter(2) and his co-workers, and also by Leonard Hill and
J.J. R. Macleod (3 and +), have demonstrated beyond question that the ill
results observed in caisson workers and divers are to be attributed entirely to
injudicious rapidity of decompression.
Experiments on animals have shown that every 100 c.c. of blood or tissue
fluid dissolve, at body temperature, about 1 c.c. of nitrogen under one
atmosphere of air; 2 cc. under two atmospheres; 3 cc. under three
atmospheres, and so on (Hill and Macleod, Hill and Ham). (5)*
This nitrogen is set free as bubbles in the capillaries and tissue spaces when
the decompression period is made too short, and by the embolism of some
vessel, may produce symptoms varying in kind and severity.
One of us (L. H.) having determined, by numerous experiments on animals,
that no ill effects follow exposure to pressures up to +seven atmospheres, if
20 minutes be allowed to each atmosphere for decompression, we determined
to investigate the effects of high pressures of air upon ourselves.
The records of caisson works and the operations of deep sea divers show
that owing to the rapid rates of decompression at present employed by
engineers and divers, very great risk is incurred by workers in caissons at
pressures of +3 atmospheres, and by divers at depths of from 100 to 150 feet.
As, however, divers usually stop a very brief time, while caisson workers
outstay a shift of from 2 to 4 hours, the body fluids of the latter become
saturated with nitrogen, hence their greater danger at lower pressures.
The limit for practical diving work is fixed by the great increase of
mortality and illness which occurs at depths much exceeding 100 feet, while
at less depths than this, accidents are by no means infrequent; being
occasionally very severe or fatal in character.
The Admiralty set 120 feet as the limit of work for their divers, while the
most daring pearl and sponge fishers sometimes reach depths of 145 feet; in
* Bohr (‘ Nagel’s Handb. d. Physiologie,’ 1905, vol. 1, p. 117) gives the coefficient of
absorption of arterial blood exposed to an atmosphere of N,, at body temperature as 1°26.
The Influence of Increased Barometric Pressure on Man. 443
this latter group accidents are numerous. Lambert, the famous diver
employed by Messrs. Siebe and Gorman, salved £100,000 at a depth of about
160 feet. On each descent he passed about 20 minutes below, and about the
same time in ascending. On the last journey he stayed longer and became
affected on his return to the surface, permanently losing the power to
retain his urine. Lambert was the man who stopped the flooding of the
Severn Tunnel, going through the tunnel (dark and full of water) in a Fleuss
dress to a distance of a quarter of a mile from the shaft, and closing the
flood gates, which had been left open; his courage deserves to be recalled.
Another diver, Erostabe, salved treasure from a depth of 171 feet, and yet
another, Ridyard, from 160 feet. These three divers of Messrs. Siebe and
Gorman hold the record for successful work carried out at great depths. Two
other divers of the same firm, in order to test a patent kind of diving
apparatus, descended to 189 and 192 feet respectively. One of these divers
(Walker) tells us he was about 50 minutes over the job, taking 30 minutes to
ascend. He ascribes his immunity from accident throughout his career as a
deep diver to his habit of slow ascent. The deepest dive on record is one of
204 feet (+884 lbs. pressure); the diver who made this record died from the
effects of too rapidly mounting to the surface.
In 1894, at Bordeaux, H. Hersent,(7) an engineer in charge of caisson
works, having first experimented on animals, found three workmen willing to
submit themselves to high pressures of air. These men were enclosed in a
steel chamber, and the experiments were conducted under the observation of
a commission composed of five members of the Bordeaux Faculty of Medicine.
Two of the workmen had had previous experience of compressed air.
In one experiment the subject was compressed to +4°800 kilos. per square
centimetre (+6827 lbs. per square inch) in 35 minutes, remained under
this pressure 1 hour, and was decompressed in 2 hours 3 minutes. On
quitting the chamber the man experienced a few “ picotements,” which lasted
for half-an-hour, but no other unfavourable symptoms. In a second
experiment, a pressure of +5°000 kilos. (+7116 lbs. per square inch) was
attained, without any subsequent ill effects beyond a few “ picotements.”
Finally, the same subject was compressed to + 5-400 kilos. (+76°81 lbs.
per square inch) in 45 minutes, remained under the pressure 1 hour, and was
decompressed in 2 hours 25 minutes. The effects are recorded in these
words: “A ressenti peu de picotements, cela tient aux bains sulfureux pris
les jours précédents.” (8).
Hersent’s experiments justify his conclusion that “avec quelques précau-
tions en sus de celles qu’on prend ordinairement, les hommes peuvent étre
comprimés et décomprimés sans danger pour leur vie, et que méme leur
444 Messrs. L. Hill and M. Greenwood, Jun. [Jan. 16,
santé n’est pas menacée quand on atteint des pressions allant jusqu’a
5 kg. 400.” (9)
Hersent and his medical colleagues do not appear to have entered the
pressure chamber themselves, so that we are not in possession of an accurate
record of the subjective effects as noted by trained scientific observers. One
of our objects therefore has been to study in detail the subjective and
physiological changes mduced by greatly increased barometric pressures ;
another object has been the investigation of the respiratory exchange under
the same conditions. In the present memoir we shall communicate the
results already obtained.
Part I.
Our experiments have been carried out in a steel cylinder kindly placed
at our disposal by Messrs. Siebe and Gorman, the eminent firm of naval
engineers, to whom we are further indebted for much valuable assistance.
This cylinder (vide photograph, p. 446) had a capacity of 42:2 cubic feet,
and was provided with a mattress, blanket, and pillows, enabling the subject
to adopt a comfortable attitude. Compression was effected by means of
a two-cylinder motor-driven pump, which could raise the pressure to
+6 atmospheres in about 40 minutes. Two decompression taps were
provided, with fine bores, permitting very careful adjustment of the rate.
of escape. The chamber was also fitted with electric light, bell, telephone,
and a thick glass observation window; the latter, however, was subsequently
covered with a steel shutter for greater security. The pressure was measured
by a Bourdon spring gauge, which had been tested for correctness. We shall
now give an account of a typical experiment. The description 1s reproduced
from notes taken at the time :—
Experiment II, 29.11.05.
The subject* (M. G.) entered the chamber at 10.40 a.m. In order to avoid
any accumulation of CO2, a constant ventilation at the rate of 25 litres per
minute was maintained.
* The measurements, etc., etc., of the two subjects were: L. H., age 39, weight (in
clothes) 873 kilogrammes, height 1°81 metres, vital capacity 3500 c.c., tidal air 510 cc. ;
M. G., age 25, weight 53 kilogrammes, height 1°65 metres, vital capacity 4000 c.c., tidal
air 300 c.c. Both were in good physical condition.
1906.| Influence of Increased Barometric Pressure on Man. 445
Time. LSE CINEG) Os Pressure. Notes.
chamber.
10.40 A.M 57° F. +0
10.50 62 =
10.55 — +16 lbs. Voice becoming nasal and metallic.
11.5 67 =
11.20 69 + 62 lbs. Sensation of slight vertigo.
11.34 68* OZ Ti
Between 11.25 and 11.40 articulation was difficult, and the subject experienced some
trouble in making himself heard through the telephone.
11.55 — +77 lbs.
12 noon 65 =
Subject quite comfortable, voice still nasal but easier to produce and much more audible.
12.4 P.M. = + 72 lbs. j
12.10 — — Pulse, 40. Respirations, 9 per min.
12.37 64 + 52 lbs.
1.0 63 +31 ,, Voice much better.
1.20 634 — Pulse, 42.
1.51 == +0
Period of compression, 54 minutes.
Period of decompression, 2 hours 17 minutes.
On quitting the chamber some itching was perceived in both forearms,,
especially the right. In about 20 minutes neuralgic pains were felt, localised
in the radial side of the left forearm. These pains gradually increased in
intensity, spreading up the arm; then, after remaining moderately intense
for five minutes, they gradually subsided. Several minutes later (about
one hour after leaving the chamber) similar pain was experienced in the
right forearm. This however did not spread upwards, was less severe and
quickly subsided. An hour and a half after leaving the cylinder the subject
felt quite well and no subsequent ill effects resulted. As will appear later,
there is good reason to suppose that the slight discomfort present at the
conclusion of this experiment is attributable to the fact that the subject
remained almost completely at rest during decompression. We may therefore
conclude that an adult may be safely submitted to a total barometric pressure-
of at least 7 atmospheres, which is, we believe, a limit higher than any
previously reached.
In the course of our investigation the following pressures have been
attained :—
Subject, L. H. Subject, M. G.
75 lbs., once. 90 lbs., once.
60 ,, twice. 75 ,, three times.
AD), Be 602 iour ss
30 ,, four times. AU) Th i
30 ,, seven ,,
* Wet cloths were placed on the cylinder at this time.
+ This reading was verified by Mr. J. A. Craw, who was present during the whole-
course of the experiment.
[Jan. 16,
ll and M. Greenwood, Jun.
i
Messrs. L. H
446
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1906.] Influence of Increased Barometric Pressure on Man. 447
In no case have any severe after effects resulted. The maximum pressure
in our series corresponds to a water depth of 210 feet, which is 90 feet
beyond the limit fixed by the Admiralty for their divers.
Supposing the special diving bell designed by one of us (L. H.) for the
slow decompression of divers were employed, it seems quite possible that
work might be carried out safely at a depth of 210 feet. Even a greater
depth than this might be attained by an intrepid man, for the limit appears
to be fixed by the pressure at which the toxic effects of high tension oxygen
become an immediate danger.
These effects have been studied by Paul Bert, Lorrain Smith (10) and Hill
and Macleod (3). When the partial pressure of oxygen reaches 2 atmospheres
(corresponding to 10 atmospheres of air, or a depth of about 350 feet of water)
convulsions may occur in animals within 20 minutes. The limit of possible
safe working is therefore about 250 feet. Conceivably this limit might be
extended by diluting the air with nitrogen so as to lower the partial pressure
of the oxygen, but we do not claim more than that our experiments show
the safe diving depth may be increased up to 210 feet.
The responsibility of those who allow short decompression periods in
caisson works is clear; every death or case of paralysis from air embolism
must be set down to the negligence of the contractor.
Next, as to the sensations we felt under pressure: the feeling of discomfort
in the ears and deafness, due to a difference in air pressure within and
without the tympanum, is too well known to need description. Owing
probably to a catarrhal condition, we were unable to open our Eustachian
tubes by merely swallowing, and were compelled to resort to a forced
expiratory effort with mouth and nose shut, the latter being held tight by
the finger and thumb.
To one of us (L. H.) who had not practised beforehand the opening of his
Eustachian tubes, the first séance was most disturbing. The sensation of
increasing deafness and discomfort, more than discomfort, in the ears, with
no obvious cause, and the inability to gain relief by the recognised method
of swallowing, produced a feelimg of mental distress which led to his
signalling to terminate the experiment. Once having learned the method
of opening his tubes, no such trouble resulted on subsequent occasions.
As to whether one possesses any real sense of the amount of pressure,
the answer must be in the negative. V. Schrotter and his co-workers (11),
who made observations in caissons sunk in the Danube at from +0°5 to +2°65
atmospheres, say that: “ Bleibt nun der Druck stationir, verweilt die
Person auf langere Zeit unter einem bestimmten Drucke, so hort mehr oder
minder rasch, off mit einem Schlage, jegliche unangenehme Sensation im
VOL, LXXVIIL—B, 2K
448 Messrs. L. Hill and M. Greenwood, Jun. [Jan. 16,
Ohre auf, nur das Gefiihl von Dumpfheit, das Gefiihl eines vermehrten
Widerstandes im Ohre, wird in der Mehrzahl der Falle, besonders von
Ungewohnten, wahrgenommen.”
We found that all distinct sensations of pressure in the ears were relieved
immediately the pump ceased its strokes, and the pressure in the chamber
became constant. Our hearing was as acute and, in the opinion of L, H.,
more acute than normally. The signal of a tap with an iron spanner on
the outside of the chamber was, to L. H., painful in its intensity.
Apart from the feelings of nervousness at being exposed to so high a
pressure (which at times were somewhat acute, especially when we were
not engaged in analytical work), we could not detect any real sense of
pressure, and certainly noticed no abnormality in our bodily functions, with
the trifling exception of the voice. Thus during Experiment XV _ the
subject (L. H.) when at +60 lbs. wrote: “ Very nervous all through
experiment; whenever time for thought, the feelings of pressure, if any, due
to non-equilibration of ears when pressure is rising.’ During the same
experiment, when the subject learnt he was at +55 lbs., he wrote:
“Thought one was lower until told. No real sense of pressure except lip
and voice change.” In another experiment, M. G. was nearly two atmos-
pheres too low in his estimate of the pressure, while in a third experiment
made at a period when custom had lessened the nervous effect, he replied to
a question at +60 lbs., “ no sense of pressure.”
The voice changes, observed in all caisson workers, were well marked in
ourselves. The alteration is distinct at +1 atmosphere, and very marked at
+3 atmospheres. The voice has a peculiar nasal and metallic quality,
losing the individual characteristics of the speaker. Thus to L. H., when
speaking in the chamber, under pressure, his voice appeared like that of
M. G. under pressure. So close was the resemblance that L. G. could
fancy himself outside and listening to M. G. through the telephone.
At +3 atmospheres the power to whisper or whistle is almost entirely
lost. L.H., who retained the power somewhat longer than M. G., could
just make an audible whistling note at this pressure.
This loss of the fine vibratile movements of the tongue and lips, a loss
probably resulting from the damping effects of the dense air, leads to a false
sense of anesthesia in the former parts. This conception of anesthesia is
interesting, as being solely excited by a lack of normal movement.
V. Schrétter and others have laid stress on the diminished frequency of
the pulse and lowered blood pressure of caisson workers. Our observations
are not sufficiently extensive to permit of any final pronouncement; but,
so faras they go, we are unable to detect any definite change in the pulse
1906.] Influence of Increased Barometric Pressure on Man. 449
frequency. For instance, in Experiment II, M. G.’s pulse was at the rate
of 40 per minute at +70 lbs. and 42 at +63 lbs. In Experiment XIV,
it was 41 per minute, at +50 lbs., 30 at +30 lbs. 42 at +10 lbs., and
41 at +2°3 lbs. This subject’s pulse is normally slow, being rarely above
60 per minute in the sitting posture; hence although there appears to
have been a diminution in frequency, the change is not nearly so striking
as in the cases tabulated by V. Schrotter (12).* L. H. found no alteration in
his pulse-rate at +5 atmospheres.
Our observations on the blood pressure have not been at all complete.
The Hill and Barnard pocket sphygmometer, depending as it does upon a
column of air acting as an elastic spring, is not a satisfactory instrument for
high pressure work, the viscosity of the dense air lessening the excursion
of the pulse very greatly.
We came to the conclusion that it was an important matter during the
decompression to move in turn every muscle and joint of the body, and to
change one’s position frequently, so as to keep the capillary circulation active
in every part. In the brain, spinal cord, and abdominal organs this
circulation is kept active by the work of the respiratory pump. In the
limbs, muscles, fat of the back and chest, on the other hand, the movement
of the blood and lymph back to the heart depends mostly on changes of
posture and the expressive action of contracting muscles. The following
observations support these views. 5
In Experiment XIII M. G. was decompressed from +75 lbs. in 95 minutes.
During decompression he flexed and extended all the limb joints at frequent
intervals, with the exception of the knees. Subsequently pain and stiffness
were detected in the knees and nowhere else.
In Experiment XIV the same subject was decompressed from +5 atmos-
pheres in 120 minutes. During the compression all the limb joints, including
the knees, were repeatedly moved. No after effects of any kind were
experienced. A further difference between the two experiments was that
in the second a pause of about five minutes was made at each atmosphere
for analytical purposes. As in each of the experiments followed by pain
(in the case of M.G.) no such pauses occurred, it is possible, but we think
not probable, that these may also play a part in hindering the development
of after effects.
The most interesting experiment in this connection is No. XV. L. H. was
decompressed from +5 atmospheres in 105 minutes, a pause of five minutes
being made at each atmosphere. During the decompression movements of
* In Schrotter’s cases there was no direct relation between barometric pressure and
pulse frequency.
450 Messrs. L. Hill and M. Greenwood, Jun. [Jan. 16, )
the joints and muscle of the limbs and back were carried out regularly. On
emerging from the cylinder, beyond a few “picotements,’ no unpleasant
symptoms were noticed.
On the next day the subject wrote as follows: “The only place I did not
move and massage was the front of the chest, where I have plenty of
subcutaneous fat. In the evening painful places were felt in the sub-
cutaneous tissues of the anterior thoracic region; one spot under each
nipple, one across the right side of the chest about the level of the ensiform
cartilage, another above the left axilla in front, and one over the right upper
arm in front. A red or purplish rash appeared over these tender places.
They felt like a spot in which a subcutaneous injection of water has been
made. Next morning the tenderness was better but still evident, and the
rash was subsiding.”
Forty-eight hours after the experiment this purpuric rash was still
discernible, and was shown to Dr. W. Bulloch and other pathologists. An
eruption occurred in a very severe case of caisson illness seen by Heller
Mager and v. Schrotter (13). They give a plate of the eruption, which is
described in these terms: “ Haut der linken Schulter und des linken Armes
an der Aussenseite, besonders in der Gegend des Olekranon und des
diusseren Condylus sowie in der Gegend des Biceps mit lividen, blaulich-
rothen netzformig verzweigten, inselformigen Flecken bedeckt, ebensolche
auch am Handriicken.” The arm of this sufferer was much swollen and
intensely painful. These observations then show the extreme importance
of active movement and massage during decompression ; instructions should
be given to all caisson workers to perform such movements while in the
air lock.
We believe the tenderness and the rash were caused by small bubbles
embolising the vessels of the subcutaneous fat in the case of L. H. The pair
felt by M. G. was probably due to small bubbles in the nerve sheaths in the
first case, in the knee joint in the second.
Part II.
The next stage of our investigation was devoted to an inquiry as to the
changes in the percentage of alveolar CO, under the altered conditions.
We have employed the method described by Haldane and Priestley (14)
The subject breathes through a wide-bored rubber tube; after a normal
expiration he expires deeply and then closes the end of the tube with his
tongue. A sample was taken from the wide tube into Haldane’s portable
CO, analyser, and examined. A bench fitted up in our cylinder enabled the
subject to collect and examine samples with ease. It may be remarked that
1906.| Influence of Increased Barometric Pressure on Man, 451
it is necessary to replace the corks at the bottom of the water bath in
Haldane’s apparatus by well-fitting rubber ones, as the air is compressed in
the corks, which leak at high pressures. Owing to the loss of the water
jacket some of our earlier experiments were unsuccessful. Great care is
also necessary in readjusting the potash levelling tube, as when the chamber
is closed a slight fall of pressure is almost inevitable owing to escape round
the washer of the door.
Haldane and Priestley have shown that the respiration is so regulated as
to maintain a constant tension of CO, in the alveolar air, which is generally
about 5 per cent. of an atmosphere. Now supposing the metabolism to be
unaffected by changes of pressure, and the regulation of respiration to
continue the same, the amount of CO, in the alveolar air must vary inversely
as the pressure attained.
Thus if p be the percentage of CO, at normal pressure, then we should
have, at two atmospheres, p/3 per cent. of CO. in the alveolar air. It will
be seen that these conditions were almost exactly realised by us. The
following table gives the result of two typical experiments. The figures in
brackets give the percentages reduced to + 0 lb. in accordance with the
above principle. Strictly speaking, the exact height of the barometer should
have been recorded, and another correction ought to have been made for
charges of temperature in the cylinder. As there is however a necessarily
large experimental error, we think it needless to allow for these minor
differences, and have accordingly assumed the normal atmospheric pressure
to be 15 lbs. to the square inch, and neglected the temperature :—
Experiment XIV. 10.1.06. Subject, M. G.
Percentage of CO in alveolar air. Pressure.
5°3 (5'3) + 0 lbs.
0:9 (5'4) (Mean of two) +75 ,,
1:0 (5:0) +60 ,,
213 (5:0) FoR
1°8 (5:4) +30 ,,
2°7 (54) i
BA (54) ae
Hxpervment XV. 10.1.06. Subject, L. H.
Percentage of CO, in alveolar air. Pressure.
47 (4°7) + 0 lbs.
0°9 (45) . +60 ,,
0-7 and 0°8 (4:5) +75 ,,
0:95 (4:75) +60 ,,
1:2 (4°8) +45 ,,
1°8 (5°4) +30 ,,
2°5 (5:0) +15 ,,
50 (50) eiOuls
452 ~~ Mesers. L. Hill and M. Greenwood, Jun. —‘ [Jan. 16,
The next tables comprise all our results. The figures vertically beneath
one another refer to the same experiment :—
Subject, M. G.
Pressure. Alveolar percentages of CO..
lbs. |
53,54 | 5:3 55 5-7 | 536-7 |. 68-be4
8 | 3°3(5°06) | = — | — _ _ --
| | | 2-7 (5-4)
—
Ot
to
oo)
“-
fs
cS
|
|
|
16 | | 2-7 (6°58) | 2-7 (6°58) -
ws 1°8 (5°4)
1°8 (5°52)
|
he bo
© Fs
Pal Tel al exea!
eo
——
21-3 (5:2)
| 21-3 (5°38) —
1-0 (5:0)
&
her |
Lh We
_
@
Wal TS] | oes}
OK
SF
| | 0-9 (4°6) ae
75 | | — | 0°9 (5 “4)
Subject, L. H.
| Ere Alveolar percentages of CO. |
|
|
l |
Ibs. | | |
AS)
Oo
Or
&
or
fo)
>
a
oO
r—)
ES
PL AA eee eA:
AS)
Oo
BS
5
Xt
oo
boas
Ee NH
vo
a
Co on
a
on
Sa
=e
Ve
=
(or)
or
= 258 6
wa rw
bo
8
= tl i
eS
roy
oS
Hd us pr OU
NGS
is wae
HS
a
)
i
—
=~
ie,2)
e9)
=
oo
8
[ees nde ee)
Vt Lee oe
TORS io
Noo @
Ges | toa Wh deta |
————~ .
SSS
SY
I |
15 j
We think these results show so close an agreement with the theoretical
values as to support the conclusion that changes in the percentage of carbon
dioxide in the alveolar air depend solely upon the physical conditions. No
increase or decrease in the pulmonary output of CO2 occurs. Metabolism,
then, in so far as it can be determined by an investigation of the alveolar air,
is not affected by increasing the barometric pressure. It is scarcely neces-
sary to add that this criterion is by no means adequate to sustain the jinal
conclusion that metabolism is, in fact, unaltered by the atmospheric condi-
1906.] Influence of Increased Barometric Pressure on Man. 458
tions; so far as it goes, however, it is in favour of such an inference.
Summing up the results of the present investigation :—
It is proved that—
(1) A man can be submitted to a total pressure of seven atmospheres
without untoward effects, provided decompression be affected gradually, and
the capillary circulation be aided by repeated contractions of muscles, joint
movements, and charges of posture.
(2) We have no sense of increased barometric pressure so long as the
former is constant.
It is probable—
(1) That the subjective effects of increased pressure, apart from voice
changes and lip anesthesia, depend upon psychical conditions such as
anxiety and excitement,
(2) The changes in the percentage of carbon dioxide in the alveolar air
are conditioned solely by physical variations, and not by any increase or
diminution in the respiratory metabolism.
In conclusion we would remark that we are unable to find any evidence
in support of Snell’s (15) opinion, that the presence of CO, in the respired
air exercises a peculiarly unfavourable influence under increased pressure.
Thus in one experiment the percentage of CO. in the chamber air, at
+ 31 lbs. was 0°62 (equivalent to over 1°8 per cent. at + 0), and no
untoward results occurred on decompression.
These researches were carried out with the aid of a grant from the Royal
Society Government Grant.
REFERENCES.
1. Paul Bert. ‘La Pression barométrique.’ Paris, Masson et Cie., 1878.
Heller Mager and v. Schrétter. ‘ Luftdruck-Erkrankungen mit besonderer Beriick-
sichtigung der sogenannten Caisson-Krankheit.’ Wien, 1900; also v. Schrotter,
‘Der Sauerstoff in der Prophylaxe und Therapie,’ etc. Berlin, Hirschwald, 1904.
3. Hilland Macleod. ‘ Journal of Physiology,’ vol. 29 (1903), No. 6.
4, —— ‘Journal of Hygiene,’ vol. 3, No. 4; ‘Roy. Soc. Proc.,’ vol. 70, p. 455.
5. Hilland Ham. ‘ Physiol. Soc. Proe.,’ July 1, 1905, p. vi.
6
7
Lo
Heller Mager and v. Schrétter. Op. cit., pp. 474, et seq.
H. Hersent. ‘Note sur ’Emploi del Aircomprimé pour l’Exécution des Ouvrages
hydrauliques, et spécialement des Fondations.’ Paris, Imprimerie Chaux, 1895.
8. —— Op. cit., p. 34.
Op. cit., p. 21.
10. Lorrain Smith. “ Pathological Effects due to increased Oxygen Tension,” ‘ Journal
of Physiology,’ vol. 24, p. 19 (1899).
11. Heller Mager and v, Schrétter. Op. cit., p. 624.
Op. cit., p. 658.
Op. cit., p. 524 and Plate ITI.
14, Haldane and Priestley. ‘Journal of Physiology,’ May, 1905.
15. Snell. ‘Compressed Air IIness, or so-called Caisson Disease.’ London, 1896, Lewis,
p. 212.
Studies on Enzyme Action—Inpase. .
By Dr. Maurice NicLovux.
(Communicated by Professor W. D. Halliburton, F.R.S. Received J ee 16,—
Read February 1, 1906.)
Ina recent number of the ‘ Proceedings of the Royal Society ’* Dr. Henry G.
Armstrong published a paper with the above title. I beg leave to draw
attention to the workt I have performed on the saponification of fats by
castor-oil seeds, and, without entering into detail, to state my general
conclusions. These are as follows :—
(a) By mechanical means it is possible to separate the cytoplasm of the
eastor-oil seeds from all the other cellular elements, particularly from the
aleurone grains.
(6) Pure cytoplasm prepared as above alone has the property of hydro-
lising fats ; its power is considerable.
(c) It acts on the fats in the same way as an enzyme, and follows all the
laws of enzyme action.
(d) Nevertheless the active substance of which cytoplasm is but probably
the support is not an enzyme; this substance, which I proposed to call
“ lipaseidine,” is destroyed by water as soon as it is no longer protected by
fats.
(ec) It is possible to repeat in vitro with isolated cytoplasm hydrolysis of
the fatty matter such as occurs in the seed at the time of germination.
* “Roy. Soc. Proc.,’ B, vol. 76, p. 606.
+ These were published in a series of notes in the ‘Comptes Rendus de |’Academie des
Sciences’ :—“ Sur un procédé d’isolement des substances cytoplasmiques,” ‘Compt. Rend.,’
1904, vol. 138, p. 1112 ; “Sur le pouvoir saponifiant de la graine de ricin,” ‘Compt. Rend.,’
1904, vol. 138, p. 1175; “Etude de Vaction lipolytique du cytoplasma de la graine de
ricin,” ‘Compt. Rend.,’ 1904, vol. 138, p. 1288 ; “ La propriété lipolytique du cytoplasma
de la graine de ricin n’est pas due 4 un ferment soluble,” ‘Compt. Rend.,’ 1904, vol. 138,
p. 1352; “Mécanisme d’action du cytoplasma (lipaseidine) dans la graine en voie de
germination ; realisation synthétique de ce mécanisme,” ‘Compt. Rend.,’ 1904, vol. 139,
p- 148; and later in a general memoir, “La saponification des corps gras,’ ‘Revue
Générale des Sciences,’ 16¢me Année, No. 23, 15 Decembre, 1905, pp. 1029—1037.
4595
On the Function of Silica in the Nutrition of Cereals.—Part I.
3y A. D. Hatt and C. G. T. Morison.
(Communicated by Professor H. E. Armstrong, F.R.S. Received December 22,
1905,—Read February 1, 1906.)
(From the Lawes Agricultural Trust Committee.)
1. Introduction.
The presence of silica in plants was first demonstrated by the analyses
of De Saussure,* who pointed out that the Graminee were particularly
distinguished by the large proportion of this constituent present in their
ash. Liebig, who classified plants as “silica plants,” “lime plants,’ and
“potash plants” according to the predominance of one or other of these
constituents in their ash, in accordance with his “mineral theory,” regarded
the silica as a necessary element in plant nutrition. This view led Wayt+
to introduce as a cereal manure a rocky material derived from the Upper
Greensand near Farnham, which contained a considerable proportion of
silicate easily soluble in acids. But when Sachs{ succeeded in maturing
maize plants in water cultures containing no silica, whereby the proportion
of silica in the ash of the mature plant was reduced from the normal 20 per
cent. or so to as little as 0-7 per cent., it became evident that silica could
no longer be placed in the same category as phosphoric acid and potash as
essential elements of plant nutrition, and Jodin§ raised four successive
generations of maize in water cultures without any supply of silica beyond
that contained in the original seed.
Other investigators again showed that the stiffness of cereal straw,
which had been attributed to the presence of silica, depends on the develop-
ment of the internodes under the influence of such factors as illumination
and exposure.
Henceforward little or no importance seems to have been attached to the
presence of silica, yet, as the following ash analyses show, it forms a constant
and considerable proportion in the ashes of certain plants, though it is
almost absent from the majority.
* “Recherches sur la Végétation,’ Paris, 1804.
+ ‘Roy. Agric. Sor. Journ.,’ vol. 14, 1853, p. 225.
t ‘Flora, 1862, p. 52.
§ ‘Ann. Agron.,’ vol. 9, 1883, p. 385.
VOL. LXXVII.—B. 24
456 Messrs. A. D. Hall and C. G. T. Morison. [Dee. 22,
Table I—Percentage of Silica in Ash.
per per
| cent. | cent:
| |
Wheat straw (Rothamsted mean)...! 62°1 |} Hops, leaves (Wolff, mean) 21 ‘1
» grain 4 Ola », cones is 17-2
Barley straw , -| 46-0 || Beech leaves i .| &1°0
>» grain a ...| 18°3 |} Larch needles ‘ 22 °5
Oat straw (Wolff, mean)... 46-1 || Calamus Rotang (Wolff, 1 anal.)...) 68-0 |
» grain Py ...| 86°3 || Bambusa arundinacea Pe ...| 28°3
Rye grass (Lolium perenne) ,, .| 26°7 || Sphagnum pulustre . .| 61°8
Maize (whole plant) . .| 430 |} Pteris aquilina 3 .| 43-7
Sugar cane a 56:4 || Hquisetum arvense A -| 41 °7
Erica Tetralix “n 48 °4
(
Owing to the inevitable presence of external dust and dirt upon plant
material before analysis, it is almost impossible to say whether the small
amounts of silica found in the ashes of many other plants are accidental or
inherent.
But while it has been demonstrated that silica is not essential to the
nutrition even of the cereals, it is hardly likely that a material present
to the extent of 60 per cent. of the mineral constituents, as in the ash
of wheat-straw, can be wholly without use in the economy of the plant.
The only experiments, however, which throw any light on its function
appear to be those of Wolff and Kreutzhage.* These investigators grew
oats in culture solutions of the type usually described as complete, but
further divided into three series, receiving soluble silica in considerable
quantity, in a small quantity, and not at all. They observed that while the
total growth was not much increased by the presence of silica, the pro-
portion of grain formed was considerably raised, a precisely similar effect
to that brought about by an addition of phosphoric acid to culture solutions
deficient in that element. Hence they concluded that the action of silica
and of phosphoric acid were in some way related, the former acting,
however, indirectly on grain formation by promoting the migration of the
food materials.
With this exception the possibility that silica plays any part in plant
nutrition appears to have been ignored, just as its practical use in the
manuring of cereals has been discontinued. Observation, however, of some
of the plots at the Rothamsted Experimental Station, which have long been
subjected to a manuring with soluble silicates, seemed to show that the
question of the function of silica required further consideration, and an
* ‘Land. Versuchsstationen,’ vol. 30, 1884, p. 161.
1905.| Function of Silica im the Nutrition of Cereals. - 457
examination of the records indicated at once that the appearances noticed
were not accidental, but had persisted from year to year.
2. Field Experiments at Rothamsted with Soluble Silicates.
At Rothamsted sodium silicate has been applied as a manure to certain
of the experimental plots over long periods of time, and shows regular and
well-marked effects.
On the permanent grass plots in the Park, which is cut for hay every
year, there are two plots receiving similar heavy applications of ammonium
salts, phosphates, and potassium, sodium, and magnesium sulphates. One
of these, which receives sodium silicate also, yields a crop exceeding by
about 10 per cent. the crop of the parallel plot without sodium silicate,
taking an average over the last 42 years. It is possible, however, that the
weakly-held sodium base has some part in this action, by neutralising
the acidity produced in the soil by the continued use of ammonium salts.
This difficulty of interpretation does not, however, apply to the barley plots.
In Hoos field, on which barley has grown every year since 1852, one
series of plots receives sodium nitrate with various combinations of mineral
manures, so as to provide plots receiving (1) nitrogen alone; (2) nitrogen
and phosphoric acid without potash; (3) nitrogen and potash without
phosphoric acid; and (4) a complete manure.
Since 1864 one-half of each of these plots has been cross-dressed with
sodium silicate; hence the effect of the silicate is seen in conjunction with
each of the elements of a complete manure. The average results obtained
are set out in Table IT.
Table IT.
| | Manures per acre. | Average over 41 years, 1864—i904.
ia E
| | Grai | St
Plot. ; rain. raw.
Sodium ee Potas- | Sodium | Magne:
itrate. | Pnos- aren |sulphate| lees, sue. . . :
| m phate. |sulphate.|°"P sulphate. Without} With |Without} With
| | silicate. | silicate. | silicate. | silicate. |
i | | |
para Posse
Ib. ewt. | Ib Ib lb. | Bushels.|Bushels.| cwt. | ewt. |
er ieaaio => ; — — — 27°3 33°8 | 16°2 Me} |:
[a= dey halite c(h ke cine aaa | — 42-2 | 43°5 | 24°6 | 25°8
| angi | ee | a 200 100 | 100 || 286 | 36-4 | 179 | 217
4 275 | 3°o | 200 | 100 100 41 °2 44°53 25°3 27 °6
In this case only a normal amount of nitrogen is supplied in the form
of sodium nitrate, a neutral salt, so that there is no acid to be neutralised
2L 2
458 Messrs. A. D. Hall and C. G. T. Morison. __[ Dee. 22,
by the soda of the sodium silicate. The beneficial effect of the sodium
silicate is chiefly shown on Plots 1 and 3, and there is little gained by its
use on Plots 2 and 4. Now Plot 3 is abundantly supplied with alkaline
salts in the shape of sodium nitrate and sulphates of sodium, potassium,
and magnesium, so the addition of a further supply of sodium in sodium
silicate would not be likely to produce any effect. Rather, if the sodium
were the active constituent, would its effect be seen on Plot 2, which
receives no alkaline salts beyond the sodium in the sodium nitrate common
to all the plots. The notable fact is that the effect of the sodium silicate
is seen only on the two Plots 1 and 3 suffering from phosphoric acid
starvation, because they have been cropped for so many years without
the application of any phosphates. The silica, in fact, would seem to
partially replace or to do the work -of the superphosphate supplied to
Plots 2 and 4.
Such an opinion, derived from the yield, may be confirmed by an examina-
tion of the plots when approaching ripeness. The most striking feature
at that time is the deferred maturity of the barley on the plots without
phosphoric acid; they remain of a greener colour, and are still erect at a
time when the barley on the normal plots has turned down and begun to
yellow for harvest. This ripening effect of phosphoric acid finds a parallel,
though to a smaller degree, on the half plots receiving sodium silicate.
On Plots 1 and 3, which are without phosphoric acid, the portions receiving
sodium silicate are always riper by a few days than the other halves which
get neither phosphoric acid nor silica.
A series of analyses of the ash of the barley grown on these plots in
1903, a wet and sunless year, and 1904, a normally warm season, also serve
to strengthen the idea that the action of the silica is in some way bound
up with that of the phosphoric acid in the plant. Table III shows the
percentages of phosphoric acid and silica in both grain and straw on the
four plots, each of which is subdivided so as to be with and without silica.
It will be seen that the lack of phosphoric acid in the manure applied to
Plots 1 and 3 is reflected in the diminished proportion of phosphoric acid in
the ash of the grain, and still more in the low percentage present in the ash
of the straw. When sodium silicate is added to the plots without phosphoric
acid the proportion of phosphoric acid in the grain ash rises, but simul-
taneously it falls in the straw ash.
On the plots receiving phosphoric acid the silicate does not always cause
an increase in the percentage of phosphoric acid in the grain ash, though as
before it generally diminishes that in the straw ash.
On all the plots the sodium silicate causes an increase of silica in the ash
1905.| Function of Silica in the Nutrition of Cereals. 459
of the grain, and particularly in that of the straw, indicating that under the
ordinary soil conditions the barley plant does not obtain all the soluble silica
it would otherwise appropriate.
Table I11—Hoos Field Barley.
Nitrogen and Pure Ash per cent. in Dry Matter, and Phosphoric Acid and
Silica in Pure Ash.
| !
| | | x
| Ni il owe Nitrogen,
Nitrogen. || pee pas Nene ene | potash, and
|| Phosphate. potash. || phosphate.
[Sea EAN AR SB PAE SSN AO) |
| With | | | | | Witt
it _ | With _ | With || | With
| ay | silica. | Ca ie, || ula | silica. | Only. | silica
ee | | | |
| || | ! |
| Sa ee ie 2s. til Sant eS. 4. | 48.
| | } |
| Grain.
i| | {
Nitrogen in dry matter...) 1°63 | 1°57 || 1°50 | 1-50 aye Seay! 1°53 | 1°54
Pure ash TAGs 198 2°27 | 2°37 ESP OG 2°36 | 2°36
Phosphoric acid in pure 35°80 | 37 “74 | 42°27 | 42°64 | 35°54 | 36°11 | 41°83 | 44°31
ash } |
Silica in pure ash ......... 14°19 | 18-67 || 16°43 | 20°60 || 15°81 | 18°00 | 16°95 | 19°71
Ratio, P,O; to N ......... | 0-38] 0-48 | 0-64] 0-67 | 0-40] O-44 | 0-64] 0
1904—
Nitrogen in dry matter...) 1°79 | 1°72 || 1°52 | 1-46 ||,1°58| 1-73 146) 1°45
Pure ash Fi 1:94} 2°09 | 2°34) 2-41 || 1:97 2°15 || 2°33 2°32
Phosphoric acid in pure 32°19 | 35°29 || 40°16 | 36-40 | 30°96 | 34°16 | 38°82 | 38°46
ash |
Silica in pure ash .........! 16°76 | 20-13 19 -62 | 20-75 || 16°45 | 17°47 |, 16°34 | 19°08
Ratio, P.O, toN ......... | 0°35 | 0-43 || 0-62] 0-60 || 0-39! 0-42 | 0-62! 0-62
Straw
1903— | HI
Nitrogen in dry TEES 0°53 | 0-43 || 0-43| 0-41 || 0-°56| 0°50 || 0-42| 0-44
Pure ash os 3°66 | 4°80 } 3-41 fe» Gs 86 4:24 | 4°82 | 3:98) 4°73
Phosphoric acid in pure 2°34) 2°40) 4:18; 3°62 2°41 | 2°19 }) 4°02 |° 4°38
ash | |
Silica in pure ash ......... | 51-98 | 63-88 | 56°67 | 64-00 || 46-22 | 55-37 || 48-14 | 57°30
Ratio, P20; to N ......... | 0-16 | 0-27 || 0-36] 0-48]! 0-18| 0-22]; 0:38] 0-48
I l
1904— i
Nitrogen in dry matter... 0°49 | 0-48 0-40 | 0-42 | 0°50 0-48 \ 0°43 0°45
Pure ash : 4°07 | 5°00} 4:36] 5-09 || 4°61 | 5-29 |) 4:19) 5-01
| Phosphoric acid m pure 2°66 | 2°13 || 4°47) 4:17 2°48 | 2:02 4-78 3°96
ash | | | j
Silica in pure ash .........| 44°00 | 52°54 | 47°19 | 51°28 || 35-91 | 44°07 \ 37°43 44°18
Ratio, P.O; toN ........- 0-21 | 0°23 0°49 | O51 |; 0°23 | 0-22 OAT O44
{ | {
As the application of a soluble silicate lowers the proportion of phosphorie
acid in the straw while raising it in the grain, it would seem at first sight to
460 Messrs. A. D. Hall and C. G. T. Morison. [Dee. 22,
act by facilitating the migration and utilisation in the grain of the initially
small store of phosphoric acid derived from the soil.
But such an interpretation of the function of silica is not borne out if the
whole amount of phosphoric acid removed by the crop from the soil on each
plot be considered, instead of the proportion of phosphoric acid in the ash.
It has already been shown that the use of sodium silicate on the no
phosphoric acid plots, 1 and 3, results in a considerable increase of crop, and
as the grain of this increased crop is somewhat richer and the straw only a.
trifle poorer in phosphoric acid than the grain and straw from the non-
silicated portions of the plots, it follows that the whole crop manured with
silica contains a greater total amount of phosphoric acid derived from the
reserves of phosphoric acid in the soil. This extra phosphoric acid derived
from the soil is itself sufficient to explain the greater yield brought about by
the silicate without attributing to the silica within the plant any specitic
action m economising the phosphoric acid there present. If the function of
the silica were to replace the phosphoric acid within the plant and enable it
to be moved off to the active tissues and used over and over again, the larger
crop due to manuring with silicates would not contain any greater amount of
phosphoric acid, but the general growth of the plant, ¢.g., the dry matter
produced and the nitrogen assimilated, would be increased. Hence the ratio
of the phosphoric acid to the dry matter and to the nitrogen would be
lowered in proportion to the increased growth, conditions which are not
realised in the cases under examination, where indeed the ratio of phosphoric
acid to nitrogen is generally slightly raised by the applications of silicate.
The results on the other hand indicate that the silicate gives the plant
such a stimulus as enables it to develop more vigorously and obtain more
phosphoric acid from the soil, and that all the consequences observed follow
from the increase of phosphoric acid thus brought about.
Wolff and Kreutzhage held that the function of the silica was to enable
the plant to make fuller use of whatever phosphoric acid it had obtained
from the soil, the Rothamsted results indicate that its action takes place
earlier, in stimulating the plant to draw more efficiently upon the vast but
dormant reserves of phosphoric acid in the soil.
3. Effect of Silica on the Development of Barley in 1904.
In order to study the question more closely it was decided in 1904 to trace
the effect of phosphoric acid and silica upon the development of the barley
on these plots at regular intervals from the time of flowering onwards. As
the effect of phosphoric acid had been most evident in forwarding the
maturation of the crop, it was considered that the later period of the growth
1905.| Function of Silica wm the Nutrition of Cereals. 461
of the crop need only be investigated, «c., the period during which the
nutrition of the plant from the soil has largely ceased aud assimilation is
coming to a standstill, while the materials previously accumulated in the
stem and leaf are migrating into the seed.
The method adopted was to take a certain number of rows of barley in the
middle of each plot and remove the whole plant, as far as possible with the
roots intact, for two yards up these rows, at weekly intervals from June 13
until harvest on August 8, or nine times in all. The plants were then air-
dried after washing the roots free from soil, the grain when formed was
separated from the straw, and both were finally dried in the steam oven, so
as to obtain the weight of dry matter ; although dealing with such small areas
it, is impossible to make more than a very approximate estimate of the yield
per unit area. The dried material was ground, and after determinations of
the nitrogen in one portion, the rest was burnt for ash, in which the pure
ash, free from sand and charcoal, and the phosphoric acid and silica were
determined.
The analytical results are set out in Appendix Tables VII to X, from
which are derived the various curves of development now to be considered
(figs. 1 to 11). Before however proceeding to a consideration of particular
cases it will be convenient to trace by means of an average result for all the
plots the general course of development in the later stages of the growth of
the barley plant.
‘Grams
June 13 20 27 July 4 IL 18, 25 Aug. 1
Fic. 1.—Dry Weights. Mean of all plots. Whole Plant and Grain.
Fig. 1 shows the mean dry weight of all the eight plots on each date, both
of the whole plant and of the grain. The crop attained its maximum weight
about July 18—25, after which it remained stationary and probably indeed
declined slightly. The result shown for August 1 is clearly exceptional; on
462 Messrs. A. D. Hall and C. G. T. Morison. [Dec. 22,
that date several of the plots happened to yield an exceptionally small
number of stems on the area harvested. ;
For the better calculation of mean results the smoothed curve also shown
in fig. 1 was drawn; by combining the smoothed dry weights read off this
curve with the true mean percentages at each date were obtained the data
contained in Table IV and expressed graphically in fig. 2.
Dry matter
2z00grams
N
510,
IN (grain)
Ioograms ,
R05 {
Dry matter
CETaIN) gy
FO; (grain)
Juneig 20 27 ~— duly Il 18 25 Aug
Fic. 2.—Dry Matter, Nitrogen, Phosphoric Acid, and Silica in Whole Plant and Grain.
Means calculated on smoothed weights of whole Plant. (SiO, on 3 scale of N and
P,O,.)
From these curves it will be seen that the dry matter of the crop goes on
increasing until about a fortnight before cutting, but the whole of the
nitrogen would appear to have entered by July 11, a fortnight before there
was any sensible grain. The phosphoric acid seems to reach its maximum at
a slightly later date, and the figures for the silica, though subject to greater
errors of determination, show that the assimilation of silica continues still
later, until the grain has progressed somewhat. Of the nitrogen within the
plant, about 63 per cent. is eventually moved into the grain and rather less
than 70 per cent. of the phosphoric acid: the migration of the phosphoric
acid does not, however, take place exactly pavi passu with that of the
nitrogen, but follows it somewhat. Of the silica but a small proportion,
9 per cent. at the maximum, reaches the grain, and nearly the whole of this
is transferred in the earlier stages of grain formation, being doubtless present
in the adherent pales and glumes and not in the seed proper.
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‘FOG Uosvag “KopreG plely SooH—' AT 14,
464 ' Messrs. A. D. Hall and C. G. T. Morison. [Dec. 22,
Perhaps the most important point brought out is that the grain establishes
a particular composition at an early stage in its development, after which,
although it continues to grow and increase in weight, it does not sensibly
alter its composition. From July 18 onwards the percentage of nitrogen and
the percentage of phosphoric acid in the grain remain approximately
constant, though the grain gains a further 50 per cent. of its weight during
the same period. Whatever chemical changes take place during the latter
stages of ripening, they consist in the rearrangement of the minerals within
the grain rather than in any progressive change in the character of the
intake.
Grams
300
pie 2
25):
es Whole
is plant
200
I
100 2
28 :
foe
i
Junera 20 27 July 4. IL 18 25 Augi 8
Fie. 3.—-Dry Weights of plots without Potash.
Grams
200 45
Whole
plant
200
3
100 a
3° VGrain
4
3
IL
June13 20 27 July 4. i 18 25 Aug. 8
Fig. 4.—Dry weights of plots with Potash.
Taking these mean figures as indicating the normal course of development
it will now be possible to review the results yielded by individual plots and
1905.| Function of Silica in the Nutrition of Cereals. 465
trace the effect of silica on the assimilation of carbon (dry matter yield),
nitrogen, and phosphoric acid, and particularly on the movement of these
materials into the grain. Figs. 3 and 4 show in graphic form the yield from
individual plots, fig. 3 deals only with Plots 1 and 2, where no potash is
supplied in the manure, while fig. 4 deals with Plots 3 and 4, each of which
receives equal amounts of sulphates of potassium, sodium and magnesium.
In each figure curves are drawn separately for the silicated and non-silicated
portions of the plot.
The accidental fluctuations in yield from week to week are too violent to
admit of smoothing, but the general character of the curves shows that
Plots 1 and 3, which receive neither phosphoric acid nor silica, give con-
sistently a much lower yield than the others. The curves representing
Plot 2 (with phosphoric acid), Plot 1 5S (with silica), and Plot 2 8 (with both
phosphoric acid and silica), do not differ from one another by more than the
extent of the accidental fluctuations from week to week of any one of them,
but all indicate a yield about half as large again as that of Plot 1. Similarly
where potash is used: Plot 3, without silica or phosphoric acid, never yields
much more than half the crop on the Plots 3S, 4, and 458, receiving either
silica or phosphoric acid, or both together. As judged then by the dry
matter produced, the silicate manuring is able to do the same work for the
plant as the phosphatic manuring on Plots 3 and 4.
Despite the magnitude of the accidental fluctuations some differences in
the character of the curves may be discerned; both Plots 1 and 3 (without
silica or phosphoric agid) reach their maximum only on August 8, whereas in
five of the other six cases where silica and phosphoric acid form part
of the manure the maximum is reached by July 18 or 25. This would
confirm the appearance in the field of deferred maturity in the absence
of either phosphoric acid or silica.
Fig. 5 shows tle proportion the grain bears to the whole plant at weekly
intervals for the four plots which receive no potash, together with the
smoothed mean of all the plots for comparison. It will at once be seen that
on Plot 1, receiving neither phosphoric acid nor silica, the proportion of
grain is below the normal, and also that the grain is later in forming. The
3 per cent. or so indicated on July 4, the earliest date when any separation
of grain was possible, would be wholly made up of the adherent pales. It
is only in the following week that the weight of grain has become sensible on
Plot 1. On Plot 2, receiving phosphoric acid, the formation of grain precedes,
and also is finally somewhat above the normal.
Plot 1S, receiving silica but not phosphoric acid, occupies an intermediate
position; though starting a little later than Plot 2, it eventually becomes
466 Messrs. A. D. Hall and C. G. T. Morison. [Dee. 22,
almost identical with it. In other words, the free supply of silica without
phosphoric acid to Plot 1S has enabled the plant to mature as high a
proportion of grain, and almost as rapidly, as does the supply of phosphoric
acid to Plot 2. The further addition of silica to phosphoric acid as on
Plot 2 S does not effect any change in the character of the development of
the grain.
40%
30%
20%
July 4 IL 18 25 Aug 8
Fic. 5.—Percentage of Grain in Plant. Plots without Potash.
30%
20%
July 4
Fie. 6.—Dry Grain in 100 Total Dry. Plots with Potash.
1905. | ‘unction of Silica in the Nutrition of Cereals. 467
In fig. 6 curves are seen representing the same succession of plots, this
time however they all receive potassic manure. Again, the proportion of
grain on the plot without either phosphoric acid or silica, 3, is low, and
its formation is retarded as compared with the normal. Plots 4 and 458,
the two plots receiving phosphoric acid, are practically identical and agree
closely with the normal, while the curve representing Plot 38, where silica
but no phosphoric acid is used, occupies an intermediate position. The
development of grain on these plots receiving potash is later than is
normal, though ultimately as high a proportion of grain to straw is
produced.
As regards the formation of grain, the curves show that phosphoric acid
hastens the formation of grain, and eventually causes a higher proportion of
the material in the plant to pass over into that state, while silica acts in the
same direction, though not to so large an extent.
25%
Junei3 20 2q July 4 II 18 25 Avert
Fic. 7.—Percentages of Nitrogen in Dry Matter.
Turning to the entry of the nitrogen, fig. 7 shows the percentage of
nitrogen in the grain and straw at the successive dates and for the four plots
receiving no potash, the mean results being also plotted for purposes of
comparison. Plot 1, receiving neither phosphoric acid nor silica, yields the
highest proportion of nitrogen in both grain and straw at each stage of the
468 Messrs. A. D. Hall and C. G. T. Morison. [Dee. 22,
growth. The use of phosphoric acid on Plot 2 reduces the percentage of
nitrogen in both grain and straw to a little lower than normal ‘level, and
this reduction is most marked in the grain. Again, silica without phosphoric
acid on Plot 1S gives rise to an intermediate curve of development, nearer
to the normal than to the curve representing the plot without either
phosphoric acid or silica. Silica added to phosphoric acid (Plot 2S compared
with 2) makes practically no difference in the curve.
July 4 Il 18 25 Aug. I 8
Fic. 8.—Nitrogen. Percentage of whole content present in the Grain. Plots without
Potash.
Fig. 8 shows the movement of the nitrogen into the grain; although both
the grain and straw of Plot 1, without phosphoric acid or silica, contain the
highest percentages of nitrogen, yet the proportion of the nitrogen within
the plant which passes over to the grain is lower on this plot than on the
normal; the transfer again begins at a somewhat later date. The phos-
phoric acid alone on Plot 2 induces both an earlier and a greater propor-
tionate transfer of nitrogen to the grain than the normal. Silica on Plot 1S
induces an earlier and more complete transfer of nitrogen, though not to the
extent caused by the phosphoric acid. On the corresponding plots with
potash (fig. 9) very similar results obtain; without phosphoric acid or
silica (Plot 3) the transfer of nitrogen to the grain lags behind the normal,
while the use of phosphoric acid (Plot 4) accelerates this process beyond the
normal, silica (Plot 3) acts in the same direction though not to the same
extent.
1905.| Function of Silica in the Nutrition of Cereals. 469
80%
60%
July 4: ria 18 25 Aug 8
Fic. 9.—Nitrogen. Percentage of whole content present in Grain. Plots with Potash.
As regards the phosphoric acid, the proportion of phosphoric acid in the
dry matter of the grain is increased by the use of phosphatic manure, as it
is also by the use of silica, especially where no phosphatic manuring takes.
place. The removal of the phosphoric acid to the grain is naturally more
complete in the cases of phosphoric acid starvation ; and when silica with-
out phosphoric acid has been supplied, almost the whole of the extra
80%
Vig. 10.— Phosphoric Acid. Percentage of whole content present in Grain. Plots:
without Potash.
470 Messrs. A. D. Hall and C. G. T. Morison. [Dee. 22,
phosphoric acid which the plant had thus been able to acquire is moved off
into the grain. This may be seen more clearly in fig. 10, which shows what
proportion of the plant’s phosphoric acid is to be found in the grain on the
successive dates. On Plot 1, without phosphoric acid or silica, the move-
ment of phosphoric acid to the grain begins much later, but is ultimately
more complete than on the normal or on the plots receiving phosphoric acid.
With silica but no phosphoric acid (Plot 1S) the migration of phosphoric
acid begins at an earlier date and the proportion transferred is much
increased, in spite of the fact that the actual amount of phosphoric acid in
the plant is also much greater than on the first plot. Exactly the same
conclusions are derived from an examination of the curves yielded by the
parallel plots receiving potash (fig. 11); the use of silica both accelerates the
migration of phosphoric acid to the grain and makes it more complete,
although a greater proportion is initially present.
duly 4 IL 18 25 Augi 8
Fig. 11.—Phosphoric Acid. Percentage of whole content present in Grain. Plots
with Potash.
The fact that a greater proportion of the phosphoric acid present in the
plant is utilised in the grain on the silica plots, seems for the first time to
indicate some specific action of the silica in facilitating the migration of
phosphoric acid, so that it is not left unused as waste material in parts of
the plant no longer active. But it will be found that the actual percentage
of phosphoric acid finally left in the dry matter of the straw is no lower
where silica has been used, on Plots 1S and 35, than on the corresponding
Plots 1 and 3 without either phosphoric acid or silica. If something like
1905.] Function of Silica in the Nutrition of Cereals. 471
0:11 per cent. of phosphoric acid be taken as the lower limit of phosphoric
acid in the straw, that limit is just as much attained in the absence as.in the
presence of silica. The greater share of the plant’s phosphoric acid trans-
ferred to the grain in the latter case comes from the fact that the amount
of phosphoric acid assimilated, though increased by the silica, is still not
sufficient for the requirements of the plant in the formation of grain, hence
the straw continues to be depleted of its phosphoric acid to the lowest limit
possible,
The consideration then of each of the factors submitted to detailed
examination—the formation of grain and the migration of nitrogen and
phosphoric acid into the grain—leads to the same general conclusion, that
an abundant supply of soluble silica renders the barley plant more able
to obtain a stock of phosphoric acid from the soil. On the plots therefore
which are suffering from phosphoric acid starvation the manuring with
sodium silicate acts like a supply of phosphoric acid; indeed, the plant does
actually thereby obtain a larger amount of phosphoric acid.
Further evidence that the silica acts by stimulating the plant to take up
phosphoric acid is derived from water cultures grown in 1904, Three plants
of barley were grown in each of four jars, holding about 3 litres of solution,
containing the following nutrient salts per litre :—
(@alctumnitrates ss cnacds.ncacnsccesccer ss 1:64 gram.
Di-hydrogen potassium phosphate... 0°29 ,,
Magnesium sulphate (crystallised)... 0°62 _,,
Rotassiumechloratenscce.cestcnccssseasens Ovjleme
with a trace of ferric chloride.
Growth was vigorous from the first; the barley plants tillered freely and
made a large number of shoots from each grain. On June 7 the nutrient
solution was replaced by distilled water, which was changed again on the 8th,
and replaced on the 9th by a fresh solution. The new solution contained
calcium nitrate, magnesium sulphate, and potassium chloride as before in all
the jars; the phosphoric acid, however, was varied as follows :—
No, 1. No phosphoric acid.
No. 2. No phosphoric acid, but 0°146 gramme silica in solution.
No. 3. 0°355 gramme phosphoric acid, no silica.
No, 4. 0°355 gramme phosphoric acid, and 0°146 gramme silica in solution.
It soon became evident that the phosphoric acid and silica, both separately
and together, had a ripening effect, which was indicated by an earlier and an
increased formation of ears,
VOL, LXXVII.—B, 2M
472 - Messrs. A. D. Hall and C. G. T. Morison. [Dee. 22,
On August 5, although the plants were by no means fully mature, it was
necessary to harvest them because of an attack of aphis. When dried they
gave the following results :—
Table V.—Barley in Water Cultures. Yield on August 5, 1904.
Dry matter.
Plot Number of Number of
ov. 5
ears. grains.
Grain. Straw and roots. Total.
grammes. grammes. grammes.
1 4 0 0 35°17 85°17
2 7 5 (0) 31°87 81 ‘87
3 18 177 5°26 50°79 56 -05
4 27 272 9 ‘63 56 °26 65 ‘89
Assuming that on June 9, when the treatment was varied, all of the plants
were approximately equal, it will be seen that the extra phosphoric acid
added to Nos. 3 and 4 allowed them to double their weight during the
remaining period of growth. The silica alone added to No. 2 did little to
enable the plant to make better use of the restricted amount of phosphoric
acid already in the plant, for although the formation of ears seems to have
been a little forwarded, the few grains that were produced possessed no
sensible weight. When, however, silica is provided in the presence of phos-
phoric acid, No. 4 compared with No. 3, it brings about a considerable increase
of growth and an accelerated formation of grain—just such a change, in fact,
as would be brought about by an increased assimilation of phosphoric acid.
In fact, these cultures demonstrate that although silica cannot replace phos-
phoric acid, nor even economise and make more effective a restricted supply
already within the plant, it will stimulate the plant to assimilate a greater
amount of phosphoric acid should: that be obtainable from the medium in
which the plant is growing. Hence, when applied to a silica plant on a soil
impoverished in phosphoric acid, it has the same effect in increasing and
accelerating the formation of fruit as would result from a direct application
of phosphoric acid.
It might be supposed that the action takes place within the soil itself, that
the sodium silicate in some way attacks the insoluble phosphates of the soil
so as to render them more available for the plant, much as an application of
lime or gypsum will liberate an increased supply of potash from the soil. On
chemical grounds it is difficult to see how such an action should occur, nor do
the results with water cultures bear out such a view. To obtain further
evidence on this point, samples of soil from the eight plots in question were
1905.] Function of Silica in the Nutrition of Cereals. 473
extracted (1) with strong hydrochloric acid and (2) with a 1 per cent.
solution of citric acid. While there is no method of determining the real
amount of plant food in the soil which is at the service of the crop, the latter
method* gives comparative estimates which are of value when dealing with
soils of the same type.
Table VI shows a series of determinations of the total phosphoric acid and
of the phosphoric acid soluble in 1 per cent. citric acid solution.
Table VI.
Phosphoric acid soluble in
Hotel phosphevieiacid. 1 per cent. citric acid.
No silica. With silica, No silica. | With silica.
0 -0086 0 :0067
0 -:0495 0:0721
0:0075 0 :0094
0 ‘0674 0 :0743
Comparing the soils with and without silica, the use of silica has not
affected the amount of total phosphoric acid; the greater draft it occasions year
by year from the soil of Plots 1 and 3, which are not supplied with phosphoric
acid, is barely visible as yet in the analyses.
The silica has also little or no effect on the phosphoric acid soluble in citric
acid on the four Plots 1,158, 3, and 38; but the amount going into solution
is distinctly higher on Plots 2S and 48 than on Plots 2 and 4, all plots
receiving phosphoric acid in the manure. It is not, however, on these plots,
but on Plots 1 and 3 that the silica shows any effect on the crop, hence these
determinations support the conclusion that the sodium silicate has no action
upon the soil phosphates.
Though the seat of the action is thus transferred from the soil to the plant,
it is by no means settled whether the stimulus which the silica gives to the
plant to enable it to take up more phosphoric acid from the soil reserves
is a general stimulus or a specific one confined to the phosphoric acid. In
other words, does the presence of a free supply of soluble silica so invigorate
the plant that it is enabled to repair any weak link in the chain of nutrition
and get as need be more nitrogen, phosphoric acid, or potash from the soil,
or is the beneficial effect confined to the phosphoric acid alone? It is chiefly
towards the settlement of this point that the further experiments both with
silica and non-silica plants are now being directed.
* Dyer, ‘Chem. Soe. Trans.,’ vol. 65, 1894, p. 115.
2M 2
A474 Messrs. A. D. Hall and C. G. T. Morison. [Dec. 22,
The further question of the intimate mechanism by which the silica acts
within the plant, and the nature of the chemical changes into which it enters
to bring about the observed effects, cannot yet be raised. In the first place
little is known of how the phosphoric acid itself acts; it is evident that it
induces seed-formation and hastens maturity, but in what way it takes part
in the cell processes is still doubtful. Some of the data accumulated in the
present investigation may profitably bear discussion in this connection—
it is evident, for example, that there is little or no interdependence between
the phosphoric acid and the assimilation or migration of nitrogen, as has often
been suggested. Again, the results would seem to indicate that a distinction
must be drawn between physiological maturity and ripeness, The grains
of a phosphoric acid starved Plot like No, 1 go through a ripening process,
but they never approach to the composition, or even attain the appearance,
of the truly mature grain on more normal Plots like 2 and 4, The grain
from Plots 1 and 3, though ripe, has still many of the characters of immature
grain, Ifthe progress of the grain be judged by such factors as the per-
centage of nitrogen or the ratio of phosphoric acid to nitrogen, the grain
early in its formation settles down to a standard composition correlated
with the original supply of nutriment, and after this point has been reached
it does not change its gross composition, though it is continually increasing
in size and weight. For example, the grain of Plot.1, with its high
percentage of nitrogen and low ratio of phosphoric acid to nitrogen, which
might be taken as indicative of its generally immature character, shows no
tendency as its grows and ripens to approximate in composition to the
thoroughly mature grain of Plot 2. The later stages of ripening are without
doubt attended by changes in the nature of hoth the carbohydrate and the
proteid contents of the grain, which however are not apparent in the
elementary analysis of the grain.
Conclusions.
The following general conclusions have been reached in the course of this
investigation :—
(1) Silica, though not an essential constituent of plant food, does play a
part in the nutrition of cereal plants, like barley, which contain normally
a considerable proportion of silica in their ash.
(2) The effect of a free supply of soluble silica manifests itself in an
increased and earlier formation of grain, and is thus similar to the effect of
phosphoric acid.
(3) The silica acts by causing an increased assimilation of phosphoric
1905.] Function of Silica inthe Nutrition of Cereals. 475
acid by the plant, to which phosphoric acid the observed effects are due.
There is no evidence that the silica within the plant causes a more
| thorough utilisation of the phosphoric acid that has already been assimilated,
or itself promotes the migration of food materials from the straw to
the grain.
(4) The seat of the action is within the plant and not in the soil.
APPENDIX.
Table VII.—Hoos Field Barley, 1904.
Actual Dry Weights of Grain and Total Plant.
No silica. With silica.
Date | a
of sample. | Plot1.'; Plot 2. | Plot 3. | Plot 18.| Plot 2S.| Plot 38.
Nitrogen | No No ete Nitrogen No No é lot ce AE DO:
only. | potash. phosphate. Baal only. potash. |phosphate.| ~° Death
}
Whole Plant.
grammes. | grammes. | grammes. | grammes. || grammes. | grammes.| grammes. grammes.; grammes. |
June 13...) 84°5 134,°5 53°1 135 °3 140 -4 101 °5 109-4 | 146°7 | 113-2
«2D bod) TRO) 187 ‘5 70°71 180 ‘6 163 ‘0 186 ‘3 146°9 | 164°9 1514
ed 107 ‘9 218 °5 96 °5 186 °3 184-7 216 4, 195 +1 202 -0 175°9 |
July 4 116 °5 236 °5 103 ‘0 201-1 194-0 203 ‘8 193 -7 184 ‘7 179-2 |
= ilo) USS) 225-0 1040 244, °5 226-6 280-7 194 ‘9 258 -0 209-1 |
oy UG) Te 2151 134-7 266 -9 241 +4, 304-0 212-2 255 ‘6 219 °3 -
on eB 161 *4 279 -2 139-3 263 °1 245 °1 219-5 260 -2 260-0 228 -5
Aug. 1 158 9 260 ‘9 111 ‘1 226 -5 223 -0 244, °8 139 °4 214 °3 197 -4
me. 8 173 °3 270-2 154 °4 217-1 +|| 211-4 249 -9 248 -7 301°1 || 228°3
Grain.
July 4... 3:2 15°8 12°5 16 *4 7-6 14°6 || 11°5
op dlced| TB 41.°7 29°5 49 *6 18-9 41°4 || 29°5
eelSes| 262 71:0 679 85 ‘0 40 °3 69 °1 55 °6
» 25...) 50°4 87-7 84-0 70°6 80 °5 85:4 || 74:9
Aug. 1...) 53°8 83 °3 88 °3 94,°9 48 -7 (oe 72-4,
Fs mpiSs 63 °8 74, °2 80 *4 95-6 92-7 108 -0 83 °9
476 Messrs. A. D. Hall and C. G. T. Morison. [Dec. 22,
Table VIII.—Hoos Field Barley. Season 1904.
Percentage of Nitrogen in the Dry Matter.
No silica. With silica.
Date :
of sample.| Plot 1. | Plot 2. Plot 3. Plot 18. | Plot 28.| Plot 38.
Nitrogen No No ene Nitrogen No No ae lot ae
only. potash. | phosphate. oe only. potash. | phosphate. or
In Grain.
July 4. 2 °245 2133 2 358 2 °228 2225 2-193 2 547 2-165
op able 2-198 I 604 2-009 1 “742 1-963 1-670 2-242 1-743
oy lls 1-735 1-431 1°715 1-516 1 ‘637 1 -460 1-739 1 -480
5 ocala S78) 1-451 1 ‘687 1-420 1 566 1-416 1 ‘677 1 504
Aug. 1. 1-944 1 ‘561 1 560 1-467 1-762 1 -430 1-747 1-424
fasts) 1-791 1 ‘517 1-578 1-461 1°716 1-463 1-733 1-448
In Straw and Roots.
June 18...| 2:039 1 °421 2 404 1 627 1 ‘524 1-604 1524 1-579
>», 20...) 1°541 1 309 1-551 1 268 1 398 1-234 1 °465 1°413
py PMlece|| Ib BEKO) 1 ‘031 1-261 1-016 1-133 1:012 1-165 1-063
July 4...| 1°162 0°797 1°151 0 ‘977 0 :957 0-909 17121 0-812
» 1...) 0°943 0 646 0-863 0 ‘658 0 ‘796 0-655 0 *862 0 ‘703
>» 18...| 0°868 0 ‘617 0 °824 0 °535 0-704 0-580 0 “747 0 624
» 25...| 0-709 0-544 0 ‘736 0 542 0 599 0 ‘591 0 ‘661 0 546
Aug. 1...| 0-687 0-498 0 626 0 °476 0 626 0 *44.4, 0 640 0 ‘537
» 8...| 0°633 0-472 0 ‘573 0-528 0 538 0-467 0 °542 0 °533
Table [X.—Hoos Field Barley. Season 1904.
Percentage of Phosphoric Acid in Dry Matter.
No silica. With silica.
Date
of sample.| Plot 1. Plot 2. Plot 3. Plot 18. |} Plot 2S.} Plot 3S.
Nitrogen No No Ghani Nitrogen No No a a2 Pi
only. potash. | phosphate, | ~°™P'€ve- only. potash. | phosphate. a
In Grain. ,
July 4...) 0-591 | 1:064 | 0-746 | 0-869 || 0-861 | 1-092 } 1°116 | 1-007
» ll..| 0-908 | 0-787 | 0:872 || 0-946 | 0-932 | 0-932 | 0-911
» 18...| 0°403 | 0°889 | 0-736 | 0:857 || 0-814 | 0-937 |. 0-889 | 0-893.
| 4, 25..., 0°572 | 0-923 | 0-693 | 0-962 || 0-845 | 1:007'| 0-879 | 0-959
Aug. 1... 0°631 | 0°945 | 0-591 | 0898 || 0-669 | 0872 | 0-827 | 0-928
» 8...| 0°650 | 0-972 | 0:638 | 0-990 || 0-776 | 0-961 | 0-820 | 0-942
In Straw and Roots.
| June 13...| 0°394 | 0-700 | 0-369 | 0-776 || 0-613 | 0°887 | 0°555 | 0-835
» 20...| 0°362 | 0-667 | 0-386 | 0-713 || 0-576 | 0-692 | 0-490 | 0-739
» 27..| 0°306 | 0-586 | 0-308 | 0-553 || 0-442 | 0°603 | 0-388 | 0°556
July 4... 0:256 | 0-543 | 0:294 | 0-511 || 0-401 | 0-654 | 0:342 | 0°545
» 11...) 0°230 | 0-497 | 0-261 | 0-483 || 0-356 | 0-483 | 0-301 | 0-474
» 18..., 0°180 | 0-404 | 0-173 | 0-364 || 0-193 | 0-416 | 0:249 | 0:392
» 25... 0123 | 0°352 | 0-140 | 0-379 || 0-163 | O-416 | 0-161 | 0-408
Aug. 1..., 0°120 | 0-317 | 0-121 | 0-308 || 0-112 | 0-294 | 0-165 | 0°317
HlBets| 20-105 s| '90)-268)m NNO 12275) 032204100116 | mOL308 0-134 | 0°364
1905.] Function of Silica in the Nutrition of Cereals. 477
Table X.—Hoos Field Barley. Season 1904.
Percentage of Silica in Dry Matter.
No silica. With silica.
Date |
of sample.| Plot 1. Plot 2. Plot 3. Plot 18. | Plot 2S.|} Plot 38. :
Nitrogen No No re Nitrogen No No za oe cre
only. potash. | phosphate. | ~O™P'°*|| only. potash. | phosphate. ae
In Grain.
July 4.... 0°331 0 920 0 °344 0 :730 0 ‘876 1-216 0 °657 1 +236
a5 aba _— 0 ‘680 1-016 0-620 0-800 0°712 0 ‘696 0°777
5 alae CORRS 0 442 0 ‘501 0 621 0-466 0 ‘631 0 -486 0-630
“5 25...| 0-294 0°329 0 448 0-336 0 -484 0 536 0 °384 0-493
Aug. 1...) 0°301 0-403 0 °382 0 :388 0-433 0-521 0 337 0 558
5 ee | 0 °339 0°475 0-339 0°417 0 443 0 548 0 °419 0-468
In Straw and Roots.
June 13...{ 1°541 1 523 1-619 1°516 1-850 2-686 1-937 1-969
» 20...) 1°506 1°515 1 ‘764 1 ‘596 2161 2-487 1°932 1 843
op 2Xloell 1724 1 502 1-299 1-259 1-762 2°391 1-881 2 °084
July 4...) 1°191 1 528 1368 0-907 1-988 2 °784 1-767 2 '257
» Ll...| 1°415 1:974 1 604 1 °578 2 516 2-527 2124 2, :280
» 18...| 1643 1 ‘966 1-404 1 569 2° 656 2-905 2 °388 2 558
» 20... 1°809 2-214 2°085 1-954 2-678 3 069 2-462 3 °291
Aug. 1...) 2°210 2 °203 1-858 1°915 2-985 3°550 2 644 3°171
5) Shoal) aL Sai 1-690 2 °356 2 °033 3-028 3 874 2 °748 2-923
478
On Innervation of Antagonistic Muscles. Ninth Note.—Successive
Spinal Induction.
By C. 8S. SHERRINGTON, F.R.S.
(Received January 31,—Read February 15, 1906.)
(Physiology Laboratory, University of Liverpool.)
It was previously* pointed out that in various reflex reactions inhibition is
succeeded by marked exaltation of activity in the arcs inhibited. This after-
effect may be figured as a sort of rebound from inhibition. -
An example is the following. When a dog in which the spinal cord ts
been transected in the thoracic region is, the period of shock having “passed,
supported so that its spine is vertical and its hind limbs hang freely, these
latter begin to perform a rhythmic stepping movement. This is the reflex,
termed by Goltz the mark-time reflex.. The tempo of this stepping differs, in
my experience, in different dogs and at different times in the same dog. It
may be as frequent at 22 steps of each leg per 10 seconds or.as slow as seven
steps in that period. It will persist in some animals for 20 minutes at a time.
After, some minutes’ duration its amplitude usually becomes less and the
movement on the whole less regular. For the first minutes of duration it is
however regular and shows little variation.
The stimulus which excites this reflex has not been traced with exactitude.
It persists after severance of the sciatic trunk not including the hamstring
nerve. Freusbergt inclined to attribute it to afferents belonging to the
“muscular sense,” and especially to those connected with parts put under
strain in the passive attitude given to the limb under its own weight. It is
closely similar to the stepping reflex studied by Philippsont in the dog sup-
ported with spine horizontal. That it is initiated by the stretch of some tissue
above the knee and especially on the flexor aspect of the hip may be argued
from its immediate cessation when the dependent limb is supported from
drooping by lifting the lower end of the thigh slightly from underneath by a
prop placed just above the knee. Such support, in my experience, usually
causes cessation of the reflex in the unsupported (fig. 1) as well as in the
supported limb and it does not matter which of the two limbs is supported.
The main stimulus, therefore, seems bilateral in origin, and to lie above the
* “Roy. Soc. Proc.,’ B, vol. 76, p. 160.
+ ‘Pfliiger’s Archiv,’ vol. 8.
+ Heger's ‘Travaux de Laboratoire,’ Bruxelles.
On Innervation of Antagonistic Muscles. 479
knee on the flexor aspect of the hip. The attachment of small weights to the
foot has not, in my experience, increased the reflex.
In the stepping reflex obtained when the animal is supported vertically
(the “ mark-time” reflex) the movement is more pronounced at hip than at
knee and ankle. A very similar stepping reflex occurs also when the animal
is nearly supine. In this latter the movement is more marked at ankle and
knee than at hip. In this posture of the animal passive dorsi-flexion of one
ankle often excites dorsi-flexion of the opposite ankle, followed by extension
at that knee and then by plantar-flexion at that ankle.
Re AOAC \ BAN Kove Nua \ NERS LANAAAAN
MOQ AAA AAA AAA WA KAN AAA
Time tn secs.
|
|
S——
Fic. 1.—“ Mark-time ” reflex of spinal dog. The up strokes correspond with flexions of
the limb, the down strokes with extensions. For the period between the two marks
on the signal line the reflex was interrupted by taking the limb’s weight off the
fellow limb to that yielding the record, namely by supporting it under the knee. On
return of the reflex, when the limb was again allowed to hang under its own weight,
the reflex shows no increase beyond its previous activity. The small undulations
during the period of rest are due to slight swaying of the animal ; the reflex ceased
completely. Time is registered above in seconds.
There is also a stepping reflex elicitable from the spinal dog when lying on
its side and without any marked mechanical strain either of flexion or exten-
sion; this is obtained by faradisation of the skin of the opposite hind foot.*
* Sherrington, ‘Journ. of Physiol.,’ vol. 33.
480 Prof. C. 8. Sherrington. [Jan. 31,
Here the tempo of the stepping is also about 20 steps per 10 seconds, but the
stepping is strictly unilateral.
These points argue that several sources of excitation probably co-operate in
the production of the stepping reflex. An important item in the execution of
the movement of the reflex in all its forms is flexion at the hip and knee.
Suppose the “ mark-time” reflex is in regular progress and is being recorded
from one knee, ¢g., right by a thread passing thence to a pulley and light
lever, if then the other thigh (left) be gently supported from behind the knee’
the record shows that the stepping reflex usually at once ceases in the right
limb (fig. 1. The refiex ceases entirely: the small undulations on the trace
in the interval during the cessation are due to swaying of the body, partly
respiratory, in the suspended attitude). The limb during the cessation of the
reflex hangs somewhat extended. On removing the slight support from
under the left knee the “mark-time” reflex at once recommences, with flexion
in the right knee. The reflex, on recommencing after this pause, continues as
it ceased, that is, its tempo and amplitude are practically the same as before
the interruption (fig. 1).
This result contrasts with the following. Goltzand Freusberg* showed that
the “ mark-time” reflex can be cut short by a strong squeeze of the tail. In
my experience this stimulus is best applied near the root of the tail. A
light touch on the hair of the tail often increases the stepping reflex, and the
stronger the mechanical stimulus to the tail the quicker and more powerful
as a rule is the inhibition of the stepping. But the stimulus to the tail need
not be very strong in order to cause inhibition. I judge that the intensity of
the mechanical stimulus which, applied to the tail, inhibits the reflex stepping
is such that, were the condition of the animal not spinal, would constitute a
dolorous (pathic) stimulus. The tail stimulus which inhibits may, therefore,
be considered adequate for a nociceptive reaction.f
The application of this stimulus to the tail does not in any way interfere
mechanically with the stepping movement. Suppose the “mark-time” reflex
to be in regular progress and recorded as before, if then the tail stimulus be
applied the stepping reflex is almost immediately arrested, and in both
limbs. The reflex remains in abeyance while the tail stimulus is continued.
On the cessation of the latter the reflex returns, and on its return soon
shows indubitable increase in activity as compared with its activity before
the inhibitory arrest (fig. 2). The increase is chiefly seen in the amplitude
of the movement, but there is also often marked quickening of the tempo of
the rhythm. I have seen the rhythm on some occasions quickened by
* ° Pfliiger’s Archiv,’ vol. 8.
+ ‘Journ. of. Physiol.,’ vol. 30, p. 39, 1903.
1906. | On Innervation of Antagonistic Muscles. 481
30 per cent. The after-increase of the reflex may persist in evidence for
many seconds. Its decline is gradual.
UUURURURURERECURUCEUCUURRUCERERECECERUURUURURUUUURRRUUUURUCEURUUGE:
Time wn sees
Sugral
'Fia. 2.—“ Mark-time” reflex as before ; but the reflex is here interrupted by stimulation of the tail.
This
arrest, due to enhibition, is followed, after cessation of the inhibitory stimulus, by increase in amplitude and
/ slightly in frequence of the reflex. The signal registers period of application of inhibitory stimulus.
registered in seconds.
;
The arrest of the stepping reflex by tail inhibition cannot be prolonged
indefinitely. The reflex tends to return in spite of the inhibitory stimulation
when the latter is long persisted in. It is different when the stepping reflex
is arrested by lifting one knee; the reflex does not then tend to break
through the arrest, however long the latter be continued. In this form of
arrest of the reflex the arrest seems referable simply to cessation of the
stimulus which excites the reflex. In the case of arrest by tail inhibition
the arrest seems referable to a central inhibition, the peripheral stimulus, E,
Time
482 Prof. C. S. Sherrington. [Jan. 31,
excitatory of the reflex remaining in action allthe time, though unable to
produce the reflex owing to the intervening inhibition.
The after-increase which ensues, in the second form of arrest, but not in
the first, might be explicable in either of two ways. It might be due to the
continuance of the exciting stimulus, E, during the period of arrest. That
stimulus might, though unable to evoke discharge of the motor neurone
during the inhibition, yet be charging a relay apparatus in the reflex are,
and so lead to increased discharge after the inhibition was past. Or the
after-increase might proceed as a direct result from the inhibition itself,
the depressed activity of inhibition being followed by a rebound to super-
activity, and altogether apart from the continuance of any excitatory
stimulus during the inhibitory period.
To decide between these possibilities the effect of strongly stimulating the
tail when at the same time both hind limbs were supported from below was
tried. The stimulus for the stepping reflex was thus held in abeyance at the
time of and during the whole period of the intercurrent inhibition. The
result was found to be an after-increase of the stepping reflex not less
marked than in the previous cases.
It is not at first obvious what relation a stimulus to the tail bears to the
reflex of the limb. But it is often noticeable that in the “mark-time” reflex
the tail itself is alternately deflected to right and left, keeping time with the
stepping reflex. When the right limb begins to draw up in flexion, and the
left limb to straighten out in extension, the tail begins to move from the
right to the left. The tail does really therefore participate in the locomotor
reflex, of which the stepping movement is also a part. Nocuous stimuli to
the side of the tail, eg., by unipolar faradisation, evoke reflex abduction of
the tail from the side stimulated, and the organ is then usually kept abducted
for a time, just as the hind paw is drawn up and kept so for a time when
excited by similar stimulation. Moreover, such stimulation of the tail
excites reflex movement not only of the tail but of the hind limb, and the
limb’s movement is usually extension at hip and knee. The result of thisis,
that the tail stimulus can inhibit a flexion-reflex of the hind limb. If the
flexion-reflex be induced by inserting a hedgehog spine into the planta, and
if while that prolonged reflex is in progress and the limb is remaining
thoroughly flexed at hip and knee, a caudal skin-point is faradised, the imb
at once drops into the extended attitude under gravity. This occurs when
the nerves to the extensors to the hip and knee have been severed. The
tail stimulus therefore inhibits the flexors of knee and hip.
The particular mode in which the tail-stimulus comes to inhibit the
stepping reflex seems to be that it inhibits the rhythmic flexion of the hip,
1906. | On Innervation of Antagonistic Muscles. 483
which is so prominent a part of the mark-time reflex. The after-increase of
the latter reflex following on its inhibition by the tail-stimulus seems, as
shown above, a pure effect of rebound from inhibition. On the above view
it should show itself therefore chiefly in an after-increase of the flexion of
hip movement, and the graphic records of the effect show this to be the
case (fig. 2), the movement of flexion being the upward movement in the
tracing.
This inhibition of the “mark-time” reflex exemplifies therefore the
principle of the common path,* The reflex arc whose reaction is inhibited
and the reflex are which inhibits are both found when separately examined
to use the same final common path, but to different effect. The common path
in this case is the flexor neurone of the hip, and one arc uses it in a steady
depressor manner and the other in a rhythmic pressor manner, The conflict
in this case, as so often, is between a nociceptive reaction and a purely
locomotor reaction; and the former prevails as is usual,f
The after-increase consequent upon inhibition is evidently a form of
“bahnung.”t In order to distinguish it from those forms of “ bahnuny”
which ensue without previous inhibition and are therefore immediate, it
may be conveniently termed “ successive spinal induction,’ the more so as that
term draws attention to the likeness between the spinal process and certain
visual phenomena commonly designated “ induction,”
Another instance of “successive spinal induction” is the following: In
the spinal animal (cat, dog) lying supine, the knee-jerk is elicited at regular
interval by tapping the patellar tendon. If, then, the central end of the
previously severed hamstring nerve is faradised, the knee-jerks become much
less ample or quite inelicitable. The tonus of the knee-jerk muscle (vasto-
erureus) is at the same time depressed. On discontinuing the stimulation of
the hamstring nerve, the knee-jerk quickly becomes again elicitable, and soon
is more brisk and ample than prior to the intercurrent inhibition§ (tig. 3).
The tonus also returns and in some cases becomes clearly greater than prior
to the inhibition. This after-increase of the knee-jerk takes place when,
during the whole period of inhibition, the leg is by mechanical support
prevented from drooping, and thus the passive stretch of the vasto-crureus
is avoided during the inhibition. The after-increase also occurs when the
elicitation of the knee-jerk is completely remitted during the whole period
of the inhibition. The after-increase is not therefore due to any continuance
* Sherrington, ‘ Brit. Assoc. Reports,’ 1904, Address to Section I,
+ Ibid.
¢ Exner, ‘ Pfliiger’s Archiv,’ vol. 28.
§ ‘Roy. Soc. Proc.,’ B, vol. 76, p. 161.
484 Prof. C. 8. Sherrington. [Jan. 31,
of the action of exciting stimuli during the period of inhibition. It is a
“successive spinal induction” following upon inhibition, just as in the
instance previously given.
\
BESeeeeanau \_\ eaaual \\ WEEEEEEEEEEREUY AA AL VASA AAA h JS \ \. MA wl
Time in secs
Fig. 3.—Knee-jerks. The knee-jerks were elicited by taps of equal intensity delivered at intervals
signalled by a metronome. During the time marked by the signal the afferent nerve of a
flexor muscle of the knee was weakly faradised. This inhibitory stimulus depressed the
knee-jerk. After the inhibitory stimulus was discontinued the jerks increased to beyond their
amplitude prior to the inhibition ; this increase is accompanied by a tonic after-action following
each jerk. Time registered above in seconds.
In the “ scratch-reflex,” after its inhibition by the crossed extension-reflex,
or the homonymous flexion-reflex, a similar after-exaltation is sometimes
seen. Fig. 4 exemplifies such an occurrence. But the time of interruption
of the reflex has usually in my records been too short to allow much scope
for the development of successive spinal induction, and the quick tiring of
the scratch-reflex under electric excitation is unfavourable to examining it
there.
1906. | On Innervation of Antagonistic Muscles. 485
aN
Time tn-2”
Stgnal B
Stgnal A
Fic. 4.—Scratch-reflex of spinal dog. The reflex was evoked from the skin of the shoulder
by unipolar faradisation applied for a period marked by the signal line A. The refiex
soon after its commencement was inhibited by stimulating (unipolar faradisation) the
skin of the planta of the foot engaged in the scratching-reflex. The time of the
inhibitory stimulus is registered by signal line B. After cessation of the inhibitory
stimulus the scratch-reflex—its stimulus being continued throughout—returned, and
on return was more ample than before the inhibition. Time registered above in
fifths of second.
486 Prof. C. S. Sherrington. [Jan, 31,
It is easy to evoke reflex-extension of the hind limb by stimulation of the
skin of the opposite hind limb. With the spinal dog laid on its side (e.g., left)
and a thread attaching the knee of the slightly flexed right limb to a recording
lever, the delivery of a certain stimulus (eg., 15 break-shock at 30 per
second) by unipolar faradisation through a gilt needle at a skin-point of the
left foot, evokes reflex-extension at right hip and knee, If this stimulus, at
moderate and unchanged intensity, be given at regular interval (e.g., once per
minute) a series of extension reflexes of regular height and duration are
obtained. If in the course of such a series the right limb is, during one of
the 60-second intervals, thrown into strong reflex flexion (¢g., by faradisation
of the skin of its own foot and the reflex flexion be maintained for some
time, ¢.g., 40 seconds) the next extension-reflex following on the intercurrent
flexion differs from those prior to it in being more ample and more prolonged
(figs. 5 and 6), Its after-discharge is greatly increased and its latency is
sometimes diminished. If the test stimulus for the extension-reflex be
adjusted at just subliminal value, the intercurrent flexion-reflex will make it
supraliminal. The exaltation of the extension-reflex may remain perceptible
for five minutes; in the example furnished in fig. 5 it is quite recognisable
for four minutes.
The mode of production of this exaltation seems the same as that traced
above in the knee-extensor, with the knee-jerk as test-sign, and in the
stepping reflex after interruption by tail inhibition. It has been shown that
central inhibition of the extensor of the knee is part and parcel of the
homonymous flexion-reflex of the leg. In the present case, therefore,
during the intercurrent flexion-reflex, the reflex arc of extension was under
inhibitory depression. After discontinuance of that inhibition the extensor
reflex is found exalted to a degree of activity beyond that which it showed
prior to the inhibition.
A similar successive spinal induction is evident in the following. The
spinal animal (cat) being supine, the nerves of one hind limb are severed
save for the nerve to vasto-crureus. The limb is supported with femur
vertical and anticrus therefore fairly flexed at knee. The central end of the
hamstring nerve is faradised; this causes the usual reflex inhibition of
vasto-crureus, an inhibition which, if there be little or no tonus in that
muscle at the time, is difficult to detect by mere inspection, though easily
revealed by abolition of the knee-jerk, If the faradisation of the central
end of the hamstring nerve be continued for some seconds, eg., three, on its
cessation there often ensues a marked reflex extension of the knee. This
is no mere return to previously existent slight tonus; it is a fairly intense
contraction of the vasto-crureus, often sufficient to extend the knee fully
1906. | On Innervation of Antagonistic Muscles. 487
and passing off again in three or four seconds’ time, It sets in, in my
experience, not at the very moment of withdrawal of the inhibitory stimulus,
but in the course usually of the first three seconds following that with-
drawal. A tap given to the patellar tendon appears. sometimes to elicit it
when otherwise it would not ensue spontaneously.
Related to this phenomenon seems the following. When a flexion reflex
of the hind limb is by appropriate stimulation continued for a long time in
the spinal dog, the flexion tends to be broken through™* from time to time
by short-lasting explosive extensions of the limb, much resembling the
“extensor-thrust.” In all these cases the extensor arc during the fiexion-
reflex has been under prolonged inhibition, and the superactivity which
it shows under a test stimulus, the “ spontaneous” discharge which it
exhibits on relief from the inhibition, and the explosive outbreak which
it gives when the inhibitory reflex is getting fatigued, all seem to be
evidences of “successive spinal induction” supervening as a rebound after
inhibition.
The effect upon the direct flexion-reflex of an intercurrent extension
reflex is, in my experience, much less marked than the converse just
described. This may be due in part to my having used the crossed
extension-reflex and not a direct extension-reflex as the intercurrent reflex.
The crossed reflex is less potent and powerful than the uncrossed reflex.t
But the only homonymous extension-reflex of the limb available is the
“extensor-thrust,” and that is so unmanageable, and especially is so little
capable of prolongation, that it was unsuited to this purpose. However,
an influence can be traced, and in other ways than by intercurrent extension
in a series of reflex flexions. Thus, with the spinal animal vertical, the hind
limbs are taken and kept fully extended at hip, knee, and ankle; then usually,
in a short time, a strong flexion-reflex at hip and knee supervenes. Again,
if similarly one hind limb be strongly passively flexed at hip but strongly
extended at knee and kept in that posture for a short time, it is usual for any
attempt to passively extend the hip to elicit at once strong reflex contraction
of the flexors of the hip, preventing passive extension.
Nevertheless, the greater inductive effect of flexion upon extension than
of extension upon flexion as examined at the knee-joint, seems, in my
experience, marked. In regard to it one remembers that though electrical
stimulation of the afferent nerve-fibres from the flexor muscles has been
shown to inhibit the reflex contraction of the extensor,t it has not been
* ‘Journ. of Physiol.,’ vol. 34, p. 34, fig. 21, phase 3.
+ Sherrington, ‘ Brit. Assoc. Reports,’ 2bzd.
t ‘Roy. Soc. Proc.,’ vol. 52, p. 556.
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1906. | On Innervation of Antagonistic Muscles.
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Fie. 6.—Crossed extension-reflex. The reflex was being elicited regularly by short series
of break-shocks, the series being equal in intensity and duration. (In 26 nine shocks
were delivered instead of eight in the other stimulations, owing to a defect in the
rotating key.) Reflex No. 1 is the last of a series of equal reflexes thus provoked ;
both stimulus and reflex were of low intensity. After reflex No. 1 in the one-minute
interval between it and 2a, a strong flexion-reflex of the limb was excited and main-
tained for 40 seconds. The next following reflex 2a exhibits augmentation, and this
augmentation is obvious also in reflex 2b. Reflex 2d was elicited two minutes after
2b, and shows no augmentation. It is somewhat less than the reflex evoked just prior
to the intercalated flexion-reflex. The signal above registers the time, etc., of the
break-shocks used as stimuli. Time is marked below in seconds. The intensity of
the stimulus and the place of its application remained unaltered throughout the
series of observations.
JEN
490 | Prof. C. 8. Sherrington. [Jan. 31,
shown conversely that similar stimulation of the afferent fibres of the
extensor muscle (vasto-crureus) inhibits contraction of the flexor muscle.
To examine this latter point is not altogether easy, since the nerve severance
of the vasto-crureus nerve, in order to stimulate its central end, of necessity
renders impossible the maintenance, let alone the examination, of any
reflex status of that muscle. I have, however, succeeded in splitting the
nerve, and if that is done without too much damage to either half of it,
one division can be severed and dissected back into the psoas, and the
other remain preserving the knee-jerk and, more important, a fair extensor
tonus at the knee. The central end of the severed division can then be
stimulated, and its effect on the rest of the extensor muscle itself be thus
observed.
To employ electrical stimulation with as little risk of escape of current
as possible it is necessary to cut the divided nerve as low as possible, and
dissect it up into psoas as high as possible; in this way I have obtained
sometimes 5 em. length of free nerve. I have further exsected the internal
saphenous nerve right up into the psoas, and also the external division of
the muscular division of the anterior crural nerve, hoping thus to minimise
current escape from the stimulated branch of vasto-crureus nerve to those
other branches of the anterior crural. In a previous communication I
discarded effects of electrical stimnulation of the nerve of the extensor
muscle as too open to error by escape of current.* Further examination
with the above precautions makes me, however, regard the following as
reliable reflex effects obtainable by stimulation of the central end of the
vasto-crureus nerve itself: inhibition of the tonus of vasto-crureus itself,
the tonus returning to some extent immediately on cessation of the stimulus
if the stimulus be weak and brief; inhibition of the knee-jerk, contraction
of rectus femoris, especially of its upper part, and tensor vagine femoris
and psoas, dorso-flexion of ankle, some slight contraction of the hamstring
muscles, especially deep inner hamstring, and extension of opposite knee with
inhibition of its hamstring muscles. These effects obtained by faradic
excitation are also obtainable, but in slighter measure, by drawing a ligature
tight upon the central end of the nerve (mechanical stimulation). The
total effect is flexion of the homonymous and extension of the opposite limb.
There is thus no evidence that the afferent nerve-fibres from this extensor
muscle when excited in these ways inhibit contraction of the flexors,
although the afferent fibres from the knee-flexor when similarly excited do
inhibit contraction of this extensor. Reciprocal innervation is evident in
the reflex effect obtained from the afferents of each muscle, for those of
* “Roy. Soe. Proc.,’ B, vol. 76, p. 283.
1906. | On Innervation of Antagonistic Muscles. 491
each inhibit one set of muscles and excite the muscles antagonistic to the
inhibited group. But in each case the reciprocal innervation has the same
direction, namely, excitation of the flexors and inhibition of the extensors.
This relation would obviously tend to make it more facile for flexion of
the limb to successively induce extension than for extension to induce
flexion.
And another consideration has to be borne in mind. The measure to
which the intercurrent flexion-reflex exalts the following crossed extension-
reflex can be estimated in terms of the relation existing in the crossed
extension-reflex between intensity of exciting stimulus and intensity of
reflex response. A somewhat widely-expressed opinion is found in the
literature dealing with reflex action to the effect that intensity of reflex
response is relatively little determined by increase of intensity of exciting
stimulus (Wundt,* Hallstén,t Biedermann,t Baglioni§). Observations by
Merzbacher|| in the intact limb of the frog, and by Parif! in the isolated
gastrocnemius, by myself** in the scratch-reflex, and by Langendorffff in
the flexion-reflex of the tortoise, show that in some spinal reflexes at least
there can be obtained marked grading of intensity of reflex response in
conformity with grading of intensity of stimulus. In the extension-reflex
of the hind limb as obtained by stimulation of the opposite hind foot the
amplitude of the movement and its duration increase with increase of
intensity of the exciting stimulus. The relation between the intensity
of the stimulus and that of the response in this reflex is, in my experience,
somewhat different from that which obtains in several other reflexes, e¢.g., in
the direct flexion-reflex and in the scratch-reflex. The successive increments of
intensity of stimulus cause increase of the extension-reflex by fairly gradual
and regular degrees up to a certain point. Beyond that point relatively
larger increments of reflex response result from increase in intensity of
stimulation (fig. 7). This character of the ratio in this reflex between
increment of stimulus and increase of response is especially evident with the
after-discharge of the latter. In regard to the successive spinal induction
exemplified by the reflex, it is clear that if the intensity of stimulus chosen
for testing the crossed extension-reflex be near below that value at which
*
‘Untersuch. z. Mechanik d. Nerven u. Nervencentren.’
‘ Archiv f. Physiol.,’ 1885—1888.
t ‘Pfliiger’s Archiv,’ vol. 80.
§ ‘ Verworn’s Zeitschrift.’
|| ‘ Pfliiger’s Archiv.’ *
4 ‘ Archives Italiennes de Biol.’
** © Physiol. Soc. Proc.,’ March, 1904.
tt ‘Sitzungsb. d. Naturforsch. Versam.,’ 1905.
+
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1906. | On Innervation of Antagonistic Muscles. 493
further increment produces great increase of response, the exaltation induced
by an intercurrent flexion-reflex need not be very extreme to give, neverthe-
less, a very apparent and great increase in the response. A not very
extreme superactivity induced in the internal condition of the are might
suffice to give the external stimulus a value equivalent to a stimulus that
would produce a very much greater reflex response.
In the homonymous flexion-reflex the increments of reflex response
ensuing from increments of intensity of stimulus follow, in my experience,
a more regular progressive increase (fig. 8) than in the crossed extension-
reflex. There is therefore with this flexion-reflex less chance of successive
spinal induction effecting an augmentation apparently so large as with the
extension-reflex. This also has to be remembered, therefore, in contrasting
the smaller effect observed in the induction of flexion by extension than of
extension by flexion in the hind limb.
The linking together of the simpler reflexes which compose a usual reflex
eycle doubtless involves several processes; it has attracted the attention of
observers from several points of view. Loeb* has illustrated how in regard to
segmental reflexes the effect of the reflex in one segment may be to transfer
the external stimulus to another segment where it in turn excites the
reflex of that segment, and so on further. In this way the reflex sequences,
which he terms “ Ketten-reflexe,” can be compounded.
Another interesting connecting process welding simpler reflexes into more
composite is that discovered by v. Uexkiill} He has shown that a piece of
musculature, under static conditions which make it of greater length, is more
prone to excitation through the nervous arcs than it is under conditions in
which its length is less. Thus, if we suppose a pair of muscles, A and B,
which under equal activity retain the lever on which they antagonistically
operate in such a position that A is equal in length to B, and if we suppose
that a new position be given to the lever such that A is longer than B, the
neuro-muscular condition becomes altered so that A is more prone to be
excited through the nervous arcs than is B. If I represent rightly in this
way the principle arrived at by v. Uexkiill, it will be seen that in some of the
experiments mentioned in this Note and in my previous ones, the con-
ditions resemble those in which v. Uexkiill finds his principle at work.
A third process, qualified to play a part in linking together simpler
reflexes so as to form from them reflex cycles of action, seems successive
spinal imduction. It appears especially fitted to combine the successive
* * Vergleichende Gehirnphysiologie, Leipzig, 1899, p. 96.
+ ‘Zeitschrift f. Biologie,’ vol. 44.
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1906. | On Innervation of Antagonistic Muscles. 495
opposite phases of such cyclic reflexes as I have termed “ alternating,’* and
shown to be particularly characteristic of the locomotor activity of the
mammalian spinal cord. If a reflex, A, not only temporarily inhibits the
action of an antagonistic reflex, B, but also as an immediately subsequent
result induces in are of B a phase of superactivity, the central organ is in
that way predisposed for a second reflex opposite to A to occur in immediate
succession to A itself. Such an effect seems proved by the observations in
this and a preceding communication. A difficulty in applying it to the
case of an ordinary alternating reflex, ¢.g., the stepping reflex of the spinal
dog, lies in the intensity and long duration of the reactions which I have
employed in order to produce it experimentally. Such intensity and
duration certainly do not occur in the course of the alternating reflexes as
ordinarily observed. This, I think, does not exclude the likelihood that
successive spinal induction is a factor which does contribute to the
mechanism of alternating reflexes, although operating in smaller degree
than as exemplified in the intenser examples obtained under experimental
conditions and mentioned in this Note.
Addendum, March 14, 1906.
Since concluding the above I have met with marked successive induction
and rebound contraction following stimulation of the proximal end of one-half
of the split vasto-crureus nerve when the stimulus has been quite brief and
weak, «.¢., has not been detectible to the tongue-tip, and has lasted only
from 1 to 2 seconds. Starting with the knee in semi-flexion, the stimulus
has caused immediate relaxation of the vasto-crureus (inhibition), followed, on
>
“3 6.—6
7.—4
516 Dr. J. E. Lane-Claypon and Prof. E. H. Starling. [Feb.'12,
Interval of 10 days, owing to failure of supply of pregnant animals.
March 17.— 7 foetuses, etc., at 16th day.
r o1.— 5 A 26th ,,
5 23.— 8 si 22nd _ ,,
, 24.—14 i 14th ,,
3 25,—12 - 20th ,,
% 27.— 5 20th ,,
es 28.— 8 a 22nd ,,
: 29.— 5 s Qist ,,
45 30.— 7 55 U7 5,
= 31.— 4 ss 20th ,,
April 1— 9 5 13th ,,
33 sueng Ee 18th ,,
: 5 » 15th ,,
9 6.—11 3 16th ,,
is p= B cs 18th ,,
This rabbit, therefore, received the fluid extracts of the viscera of 160 foetuses. It was
killed on April 8. On reflecting the skin of the abdomen, the mammary glands were seen
to be markedly hypertrophied. The margins were almost contiguous, and were somewhat
raised and pink, presenting, therefore, much the appearance which is seen in a pregnant
rabbit of the eighth or ninth day. The general aspect of the stained gland is shown in
fig. 4. On microscopic section not only was there marked duct proliferation with mitotic
figures, but at the thickened border the formation of alveoli was just commencing. The
whole of these injections had been made intra-peritoneally, or, on one or two occasions,
under the skin of the legs, so that there was no infiltration of the connective tissue
surrounding the mammary glands. This is the best result which we obtained.
Experiment 11. May 2 to June 1, 1905.—In this experiment we sought to determine
whether the growth-producing substance is contained chiefly in the viscera or in the body,
a.e., muscles, skin, and bones, of the foetus, and, moreover, whether it could be extracted
from these tissues by boiling. Unfortunately, however, out of the four rabbits which we
chose for this experiment, only two were definitely virgin, so that the results in the other
two cases were equivocal.
Rabbit 1, not a virgin, received the pressed juice of the viscera.
Rabbit 2, a virgin, received the filtered boiled extract of viscera.
Rabbit 3, also a virgin, received the pressed juice of the bodies of the foetuses unboiled.
Rabbit 4, which was evidently multiparous, received the boiled extract of the bodies of
foetuses. From this rabbit, at the commencement of the experiment, a small portion of
mammary gland was taken as a control.
All four rabbits received portions of 182 foetuses of all ages between May 2 and May 31.
From Rabbits 1 and 4 milky fluid could be expressed from the nipples after the ninth
injection.
In Rabbit 2 a watery fluid could be expressed from the nipples after the seventeenth
injection. ;
Rabbit 3 showed traces of watery secretion after the twelfth injection.
All four were killed on June 1. Results were as follows :—
Rabbit 1.—Multiparous. Mammary glands well developed and showing many alveoli
on microscopic section. The ducts were full of milk; they were, however, lined with
-only a single Jayer of epithelium, and it was impossible to say that any hypertrophy had
taken place.
Rabbit 2.—Virgin. The mammary glands did not present much enlargement as judged
from inspection. On microscopic examination, however, many branching ducts were
1906.| Growth and Activity of the Mammary Glands. 517
observed lined with two layers of cells, presenting the same appearance, but in a smaller
degree, as those in the glands of the rabbit in Experiment 10.
Rabbit 3.—Virgin. Glands large, hypertrophied, containing a fair amount of watery
fluid. Alveoli present and ducts showing proliferation.
Rabbit 4.—Not virgin. Mammary glands fully marked, and distended with milky
fluid, but impossible to determine whether or not hypertrophied.
In order to be certain of the induction of growth in the mammary gland by the
injection of extracts of foetus, three more experiments were made. In the first of these,
in which the rabbit received 16 injections of the pressed juice of the viscera of
138 foetuses, the results were absolutely negative. In this experiment, however, we had
been obtaining very small amounts of pressed juice from the tissues, and we thought that
the absence of result might possibly be due either to retention of the active substance by
the Kieselguhr or to insufficient destruction of the cells in the process of grinding. It is
possible, too, that immaturity of the rabbit may have been in some measure responsible
for the negative result.
In the next two experiments, therefore, we abandoned the Biichner method and, after
grinding with sand and with normal salt solution, centrifuged and filtered the supernatant
liquid through a Berkefeld candle before injection. Both these experiments gave positive
results.
Experiment 12.—October 4 to 21. Virgin rabbit, full-grown. Received daily, intra-
peritoneally, the saline extract of the viscera of a number of foetuses about the fifteenth
to twentieth day of pregnancy. Killed on the 21st. It showed distinct growth of the
mammary glands with duct proliferation (vide.fig. 5).
Experiment 13.—October 4 to 21. Virgin rabbit. Received the saline extract, intra-
peritoneally, of the bodies and placentze of the same foetuses used in Experiment 12.
Fifteen injections were given in the 17 days. Killed on the 21st. It showed marked
erowth of mammary glands with plentiful mitotic figures. The appearance of this gland
in the stained specimen is shown in fig. 6.
Discussion of Results.
From the results just described, it will be seen that in six cases we
succeeded in producing in virgin rabbits a growth of mammary glands
similar to that occurring during the early stages of pregnancy, and consisting
in the proliferation of the epithelium lining the ducts, with the multiplication
of these ducts by branching into the surrounding tissues. In one of these
(Experiment 10) where our injections were carried out during five weeks
and the experiment lasted nearly seven weeks, there was an actual formation
towards the periphery of the gland of secreting acini. In some of these
cases, however, namely those in which the injections had been given under
the skin of the back (¢g., Experiment 9), the mammary glands were bathed
for considerable periods of time in the injection, and it seemed to us possible
that this might be a determining factor in producing growth.
We therefore carried out a control experiment on a virgin rabbit, in which
normal rabbit’s serum was injected, for the most part subcutaneously, for a
period of three weeks. The serum, which was derived from non-pregnant
animals, but contained much more nutrient material, ¢c.g., proteid, than the
518 Dr. J. E. Lane-Claypon and Prof. E. H. Starling. [Feb. 12,
fluids used in our previous injections, ran down in the subcutaneous tissue, so
that during the whole duration of the experiment the abdominal wall was
thickened and cedematous through the presence of the serum. On killing
the animal at the end of three weeks the glands were little, if any, larger
than those usually obtained from a virgin animal (fig. 7). On section, how-
ever, mitoses were present in the epithelium of the ducts, and there was
apparently a certain amount of proliferation of the ducts.
We must conclude, therefore, that superabundant supply of nutrient
material in the fluid surrounding the acini may lead to proliferation
resembling in kind that which was produced by our injections. ‘This result
had not been produced in an earlier control experiment, in which we injected
the saline extract of liver, and in view of the small results produced by the
injection of the serum as compared with those produced by the injection of
the extracts of fetus much poorer in proteids, we are inclined to believe that
it is impossible to explain our results in the other experiments as due to the
infiltration of the tissues round the glands. This explanation, at any rate,
could not hold for the growth in Experiment 10, in which there had been at
no time any injection into the subcutaneous tissue of the back. It is
interesting, from the general pathological point of view, to note that typical
epithelial proliferation in the ducts can be produced by an abnormally large
supply of proteid in their surrounding lymph, and the subject is worthy of
further investigation.
A striking fact in all our experiments with a positive result is the small-
ness of the growth produced as compared with the quantity of material
used for injection. In all the positive cases the material for injection was
derived from foetuses ; in Experiments 11 (2) and 12 from the viscera only ;
in Experiment 11 (3) from the bodies only; in Experiments 9 and 13 from
the foetuses together with placentz ; and in Experiment 10 from the fcetuses,
placenta, and mucous membrane of uterus together. On the other hand.
injection of extracts made from ovaries, uterus, or placenta alone had no effect
on the growth of the gland. We are therefore justified in concluding that
under normal circumstances the hormone which is responsible for the growth
of the mammary gland during pregnancy is produced mainly in the growing
embryo. This hormone, however, must be produced in minimal quantities.
It is apparently not stored up in any of the tissues of the fcetus or of the
placenta, so that, in injecting extracts of foetus, we are simply injecting the
small amount of material which is diffused through the juices on its way to
the blood-vessels and into the maternal blood.
It is possible, of course, that the specific mammary hormone is produced
from a precursor or mother-substance in some organ or other, and that future
1906.] Growth and Activity of the Mammary Glands. 519
research may reveal some method of splitting off the hormone in large
-quantities, and also of determining whether its production is diffused through-
-out all the tissues or is confined to one special organ of the body. Injection
-of extract of duodenal mucous membrane, for example, would give only
minimal effects on the pancreas. We should not be justified in concluding
from this absence of result that the duodenum was not the seat of origin of
the chemical stimulus to the pancreas. Its peculiar relation to the pancreas
is only brought into prominence when it is treated with acid, so as to liberate
the secretin from its mother substance.
Our experiments, therefore, throw no light on the seat of production of
the hormone in the feetus. Apparently the extent of the growth obtained is
-a function of the quantity of tissue used in preparing the extracts. The wide-
spread occurrence of the substance in the body of the foetus points to its
‘being extremely diffusible, as indeed we should expect from analogy with
-other hormones.
We can only say, therefore, that the hormone is produced by some or all
‘the tissues of the fertilised ovum, whence it is carried off by the blood to the
placenta, and so makes its way by diffusion into the maternal blood-vessels.
‘Whether it is identical with the substances which are responsible for the
production of the other changes associated with pregnancy, or whether there
are distinct substances acting on each organ which is modified during this
condition, our experiments do not show. But we have evidence that in the
foetus itself the hormone or hormones of pregnancy have the same result as
rin the maternal organism. Thus there is increased growth of the mammary
glands in the fcetus during the last month of pregnancy, and also in the
‘female an increase in the uterine mucous membrane, as has been shown by
‘Halban. After birth the mammary glands may begin to secrete just as after
pregnancy, and there are changes in the uterine mucous membrane similar to
‘those associated with menstruation.
Are we to regard, then, the fcetus as the only source of this hormone ?
The facts mentioned at the beginning of this paper show that such a
-conclusion is impossible. The growth of the mammary glands which occurs
-at puberty can only be ascribed to ovarian influence, and is absent if the
ovaries have been previously removed, and Halban ascribes to this ovarian
substance both the growth of the mucous membrane during each pro-cestrus
-and the swelling of the glands at each cestral period, which may in rare cases
be attended or followed by the actual formation of milk. Halban explains in
‘the same way those cases recorded by Heape and Kehrer, in which bitches,
which had not been impregnated at the normal time, have, after two months,
mot only made a bed for their young, but have had swelling of the mammary
VOL. LXXVII.—B. 2
520 Dr. J. E. Lane-Claypon and Prof. E. H. Starling. [Feb. 12,
glands, with, in some cases, actual secretion of milk. He would regard this.
condition as being a continuance of the state of pro-cstrus leading to:
continued growth of mucous membrane and also of the mammary gland.
When the impregnation was no longer possible, with the discharge of the:
ovum, the secretion of this substance ceased, and the absence of the inhibitory
stimulus caused break-down of the uterine mucous membrane as well .as.
dissimilative activity of the mammary gland.
During sexual life, therefore, the ovaries are continually producing a
substance which exerts an influence on both glands and uterus. With the
occurrence of conception there is at once a great growth of what we may
call germinal material. With the growth of the fertilised ovum the amount
of hormone produced in the ovum must also increase in proportion. In
the early stages of pregnancy the chief source of this hormone may perhaps
be located in the chorionic villi, but with the growth of the body of the
fcetus this latter must take a preponderating share in the preparation of
the hormone. We have no reason to suppose that the foetal elements of
the placenta entirely lose this function of the germinal cells, but the negative
results of injection of placente in our experiments show that it is impossible
to ascribe to the placenta, as is done by Halban, a preponderating part in
the preparation of this hormone.
If the hormone is produced in the body of the foetus, it might be objected
that the formation should go on after birth, and therefore lead in the new-
born animal to a continuance of the growth both of. the mammary gland and
of uterus. The profound changes in the environment of the new animal
which oceur at birth must, however, induce equally profound changes in its
metabolism, and there is no difficulty in imagining that with the assumption of
extra-uterine life the formation of this substance in the foetus comes to an end.*
The occurrence of growth in the mammary gland of a virgin rabbit and of
secretion in the mammary gland of a multiparous rabbit from the injection
of boiled extracts of foetus, seems to indicate that the specific hormone, like
adrenalin or secretin, is not destroyed by boiling. Further evidence,
* In the ornithorhynchus pregnancy is associated with the growth of mammary glands,
although the embryo in this animal is contained in an egg, and does not enter into any.
anatomical connection with the uterine wall. Halban points out, however, that the shell
of the egg is porous, and that during its stay in the uterine cavity it increases in size.
and the contained embryo grows, in consequence of the absorption of nutrient material’
from the fluid contained in the uterine cavity. If the embryo is able to absorb nutrient
material from the uterine contents, it is equally able to give up to these contents diffusible
substances, which may be taken up by the mucous membrane and carried by the circula-
tion to the mammary glands. The condition in the ornithorhynchus cannot therefore be
regarded as a disproof of the chemical theory which we have adopted throughout this
investigation.
1906.] Growth and Activity of the Mammary Glands. 521
however, is required on this point, as also on the question whether the
substance is specific to the animal, or whether the injection of extracts of the
foetus of one animal would produce a growth of the mammary glands in
another species. One experiment, in which we fed a kitten for three weeks
on the foetuses of rabbits, was negative in its results. This might, however,
have been due to the failure of the intestine to absorb the hormone without
destruction, or to the failure of the immature glands to react to the minute
stimulus which they received. So far as we know, secretin is not absorbed
into the circulation when introduced into the stomach or intestine, and
colossal doses of adrenalin have to be given by the mouth in order to produce
any systemic effects.
The effect of the injections of fcetal extracts on multiparous rabbits
deserves some further mention. The multiparous rabbit differs from a virgin
rabbit in possessing ready-formed alveoli, 7.v., secretory structures. On the
theory which we have adopted, the circulation of the mammary hormone
should diminish any secretion in these alveoli and should cause growth. In
all our experiments at least 24 hours elapsed between each two injections.
It is probable that the hormone was rapidly absorbed from the injection, and
was therefore present in the blood of the animal only for a certain fraction,
say a few hours, out of the 24. While it was circulating it should cause
building up of the secreting cells. Directly, however, it ceased to circulate,
the cells would enter into dissimilative activity resulting in secretion. By
our injections, therefore, we are not able to imitate the continuous stimulus of
pregnancy. We are rather producing each day a pregnancy of a few hours
followed by a parturition. These factors should therefore result in the
production of milk in any animals possessing the structures (i.c., the alveoli),
which are capable of secreting milk, and would therefore account for the
secretion of milk observed by us in all the cases where multiparous rabbits
were the object of our experiment.
CONCLUSIONS.
So far as our experiments go, they show that the growth of the mammary
glands during pregnancy is due to the action of a specific chemical stimulus
produced in the fertilised ovum. The amount of this substance increases
with the growth of the foetus, and is therefore largest during the latter half
of pregnancy. Lactation is due to the removal of this substance, which must
therefore be regarded as exerting an inhibitory infiuence on the gland cells,
hindering their secretory activity and furthering their growth. It is probable
that the specific substance is diffusible, and will withstand the boiling
temperature.
§22 Growth and Activity of the Mammary Glands,
We cannot, however, claim that these conclusions of ours are firmly
established. A final decision can only be given by a research carried on
under more favourable conditions. One requires, in fact, a farm, where we
could have at our disposal 500 rabbits, and could arrange for a plentiful
supply each day of rabbits about the middle of pregnancy. Under these
conditions it might be possible to determine both the seat and nature of the
effective stimulus, as well as to test the influence of various reagents in
splitting off the hormone from some possible precursor. Many of our
experiments, carried out in a London laboratory, were brought to a premature
conclusion by failure of material. If, however, the conception of the action
of the mammary hormone, which was put forward by Hildebrandt and adopted
by us, is correct, namely, that it is a substance which produces growth by
inhibiting the normal activity of the gland cell, it should be possible to
decide many questions affecting it by working on an animal, such as the goat,
in lactation. Injection of the hormone should diminish or stop the
secretion of milk while it was circulating in the blood, but should, as a
secondary effect, produce an increased secretion as a reaction from the
immediate assimilatory effect. The injection might, indeed, have to be
prolonged for one or two days, since we know that in Man the onset of a
renewed pregnancy during lactation stops the flow of milk only after some
time (three or four weeks). At any rate, such experiments could be more
rapidly carried out than those which have been the subject of this com-
munication.
DESCRIPTION. OF PLATE.
The drawings were made as follows:—The mammary glands were dissected out, pinned
on corked rings, hardened in corrosive sublimate and formol, washed, and stained in very
dilute hematoxylin. They were then dehydrated, cleared, and mounted as lantern slides
in canada balsam between glass plates. (These specimens were shown by projection at
the meeting of the Royal Society, on March 1, 1906.) An image of the specimens was
thrown (without magnification) on to a piece of millboard, and the darkly stained glands
were traced out in indian ink. The figures, therefore, reproduce the glands in natural
size.
Fie. 1.—Gland from virgin rabbit.
2.—Mammary gland from primiparous rabbit, five days after impregnation.
3.—Mammary gland from primiparous rabbit, nine days after impregnation.
4.—Mammary gland from virgin rabbit which had received injections of extracts
of foetuses, uterus, and placente during five weeks (Exp. 10).
5.—Mammary gland of virgin rabbit, showing growth produced by injection of
extracts of foetal viscera during a period of 17 days.
6.—Mammary gland of virgin rabbit, showing growth produced by injection of
extracts of foetal bodies and placentze over 17 days.
7.—Mammary gland of virgin rabbit, showing slight growth induced by daily
subcutaneous injection of rabbit’s serum (from non-pregnant rabbits) during
a period of three weeks.
2?
aun
Lane-Claypon and Starling. Roy. Soc. Proc., B. vol. 77, Plate
Fic. 6. Fila. 7.
523
The Internal Anatomy of Stomoxys.
By F. Tuttocu, Lieut. R.A.M. Corps.
| (Communicated by Professor E. Ray Lankester, F.R.S. Received February 2,—
Read March 1, 1906.)
The dissections of the local variety of Stomoxys, which form the subject of
this Note, were made at the suggestion of Professor Minchin, during his
direction of the Royal Society’s Commission on Sleeping Sickness in Entebbe,
Uganda. The main object was to furnish some comparison between the
internal anatomy of Stomoxys and that of Glossina, and the following notes
are based on Professor Minchin’s description of Glossina palpalis.
Complete digestion of the human trypanosome seems to occur in 48 hours,
in the alimentary canal of Stomoxys; but Lieutenant Gray, R.A.M.C.,
has found a limited percentage of these Stomoxys to be infected with a
Herpetomonas.
I am much indebted to Professor Minchin for advice and assistance at
every turn, without which these notes could not have been completed.
Digestive System.
The cesophagus emerges from the chitinous pharynx (which, with the
mouth parts, has been described by Hansen) as a flattened tube, which
gradually narrows and becomes cylindrical, running at first upwards and
then backwards to reach the brain. The connectives of the brain are more
vertical than in Glossina. On emerging from their constriction the
cesophagus dilates gradually, and runs down to enter the ventral aspect of
the proventriculus, which lies in the anterior third of the thorax.
The proventriculus (fig. 1, P.) is a mushroom-shaped viscus with a
thickened border, and lies with its convexity pointing upwards and slightly
forwards. Except for the inversion of its lateral edges, which gives to the
proventriculus of Glossina a characteristic outline, the corresponding structure
in Stomoxys is very similar in every way. The cesophagus enters the
proventriculus a little in front of the centre of its concave ventral surface,
and the duct of the sucking stomach running up from below appears to enter
with it, though in reality it enters separately at a point immediately behind.
As in Glossina, the cesophagus and the duct of the sucking stomach are
in the same line.
The thoracic intestine (fig. 1, T.I.) arises from the convex dorsal surface of
the proventriculus at a point posterior to the entrance of the cesophagus on
VOL. LXXVII.—B, 2Q
524 Lieut. F. Tulloch, [Feb. 2,
the ventral surface. From its origin the intestine runs down into the
abdomen of the fly as a narrow tube of uniform diameter, until it reaches
nearly to the lower border of the sucking stomach. At this point it dilates to
several times its former diameter, its wall at the same time becoming thinner.
The abdominal intestine is proportionately shorter, less coiled, and more
distensible than in Glossina; it is about three times as long as the fly itself.
The dilated portion of intestine has three simple coils which lie superposed
in the middle part of the abdomen, and then gradually narrows, continuing
as a uniformly narrow tube downto therectum, The narrow lower intestine
has variable bends in its course, but is not coiled.
The rectum (figs. 1 and 2, R.) is a dilated cone-shaped portion of intestine,
the apex of the cone being towards the anus. Its walls are transparent, and
through them are readily seen four long trumpet-shaped papillee, so-called
rectal glands, the narrow ends of which are inserted towards the anus
(fig. 2, R.P.). A single trachea enters the base of each “gland.” Below the
apex of the dilated cone the rectum is continued to the anus as a short
narrow tube. In the female this terminal portion of intestine runs within
the ovipositor, the anus being situated between the last segment of the
ovipositor and the terminal plate. In the male the ejaculatory duct passes
over it dorsally from left to right, and runs anteriorly to enter the penis.
The appendages of the alimentary canal are the Malpighian tubes, the
sucking stomach, and the salivary glands.
The Malpighian tubes (fig. 2, M.T., MT.) arise from the narrow lower
intestine. The proctodeeum, between their origin and the anus, comprises in
length about one-fifth of the abdominal intestine. At their point of origin
(figs. 1 and 2, O.) the intestine has a shallow linear constriction. Two tubules
arise on each side from a short common tube, and all four tubules are
approximately of the same length. The two tubules arising from one side
have thickened terminations (fig. 2, T.T.), some four times greater than a
salivary gland, and these thickened endings lie in the pericardial sinus.
The tubules of the other side are of the same thickness throughout, and
their ends lie amid the fat-body of the lower abdomen. Microscopically the
tubules are of the usual type.
The sucking stomach (fig. 1, S.S.) is a thin-walled sac, made up of one layer
of flattened cells with occasional strands of unstriped muscle. It ends at
the waist in a very fine duct (D.S.S.) which runs up ventrally to the thoracic
intestine and enters the proventriculus (P.) immediately behind the opening
of the cesophagus. The alimentary canal and the ducts in the thorax lie
in contact with each other in a narrow space between the lateral masses of
thoracic muscles. j
Cr
bo
or
1906. ] The Internal Anatomy of Stomocxys.
Fic. 1.—Alimentary Canal of Stomoxys. Dorsal view. The thoracic muscles were
removed, and the structures in the thorax separated and spread out, though
their relative positions are otherwise maintained. In the abdomen the position
of the coils of intestine has been very little disturbed, but the Malpighian
tubes have been removed by severing their common ducts on each side near O.
P., proventriculus; T.I., thoracic intestine; R., rectum; D.S.G., duct of
salivary gland ; 8.G., salivary gland; S.S., sucking stomach ; D.S.S., its duct ;
O., point of origin of Malpighian tubes; H., the dorsal blood vessel cut short
at the heart.
The salivary glands (figs. 1 and 2, S.G.) are partly thoracic and partly
abdominal. They are comparatively shorter and thicker than in Glossina.
In the abdomen they are ventral to the sucking stomach, and from a dorsal
view only a knuckle of gland is exposed at the lower border of this viscus.
2Q2
526 Lieut. F. Tulloch. » [Feb. 2,
The slightly bulbous ends of the glands lie under the upper border of the
sucking stomach, and are found by following up the outer limb of the
exposed angle of gland. Except for this angular bend the glands are straight
in their whole course, and even when pulled out they are not long enough
to reach the hinder end of the fly. .
The salivary glands run up through the waist of the fly on either side of
the duct of the sucking stomach and ventral to the intestine, and continue
with the same thickness to the front of the thorax. At this pomt, in the
neck of the fly, the glandular portion ceases abruptly, to be continued as a
very fine narrow duct (fig. 1, D.S.G.). At first this duct is made up of small
flattened cells, but it almost immediately acquires the structure of a small
trachea, becoming chitinised and having similar annular thickenings. At
the base of the brain the two ducts join and continue as a single duct on the
ventral surface of the chitinous pharynx, inside the transparent membrane
which wrapsit round. The dilatationin the common duct which Hansen has.
described and which he regards as a storage chamber for the secretion, occurs.
about half-way in the length of the common duct. The point of entrance of
the duct into the proboscis has been described by Hansen.
Nervous System,
This consists of the brain and the thoracic ganglion, with the nerves
arising from them.
The nervous system was not dissected in great detail, but the following
nerves were traced as described. The thick nerve to the ocelli arises from
the upper part of the back of the brain. The stout nerves to the antenne
arise from the front of each cerebral ganglion. On either side of the front of
the brain below the nerves to the antennz arises the slender pharyngeal
nerve trunk, which shortly divides into three. The outermost of these three
branches divides into two, one filament supplying the depressor muscle of the
pharynx which arises from the postero-superior process, andthe other running:
down inside the pharynx in close relation to its chitinous wall. The middle
division of the pharyngeal nerve joins its fellow of the opposite side on the
wall of the cesophagus as the latter enters the pharynx, the common trunk
thus formed splitting into four branches to the intrinsic muscles of the
pharynx. The innermost branch of each pharyngeal nerve joins a slender
nerve arising in the middle line. The nerve thus formed supplies the
pharyngeal muscles, but was not traced in detail.
The brain is connected with the thoracic ganglion by the connectives,
between which passes the cesophagus and which join after this to form a long
1906.] . The Internal Anatomy of Stomoxys. 527
connecting band as in Glossina. The thoracic ganglion is somewhat pear-
shaped, and is supported by the internal chitinous skeleton of the thorax,
from the surfaces of which arise the wing and leg muscles. Six pairs of
nerves arise from the thoracic ganglion and supply the thoracic muscles.
The abdominal nerve trunk continues from the posterior part of the
ganglion running down in contact with the abdominal wall. It gives off
three fine branches which supply the abdominal muscles, and ends in the
third segment of the abdomen by dividing into three. Each of these branches
again divides to supply the generative organs, the outer two running to the
ovaries or testes and the middle one to the muscles of the ovipusitor or penis.
Circulatory System.
This consists of the heart and its continuation, the thoracic aorta. The
heart is a tubular organ of the same type as in Glossina with chambers, ostia,
and alary muscles. The wall, too,is composed of similar giant cells. Though
several. stained preparations were made it was impossible, owing to the fat-
body which obscured all detail, to count the chambers and cells in the heart
wall. They seemed, from a comparison of all the preparations, to be reduced
in proportion to the smaller number (four) of abdominal segments possessed
by Stomoxys. eat
The dorsal aorta consists of paired cells, as in Glossina, and runs up on the
dorsal surface of the intestine to end on the cesophagus in a similar mass of
cells. On the surface of the proventriculus, to which it is bound down, it
becomes expanded and flattened, narrowing again to its termination.
Male Generative Organs.
These are comparatively simple. The testes (fig. 2, T.) are a pair of
smooth, oval, orange-brown bodies with a shallow equatorial constriction.
Their colour is due to a pigmented coat as in Glossina, but there is apparently
not the same tubular structure.
From the lower end of each testis arises a very fine duct (D.), short and
straight, which runs down to join the duct of the opposite side as the upper
limbs of a Y. From this junction an exceedingly short length of common
duct (C.D.) runs into the bulbous upper end of a tubular organ, which
would seem to function asa vesicula seminalis.
' This vesicula seminalis (V.S.) is a flexible tube, often. lying with two
U-shaped bends in its course. At its upper part it is bulbous, gradually
narrowing below this to end as an ejaculatory duct, which crosses the rectum
dorsally from left to right, to enter the penis in front of it; it does not thus
528 Lieut. F. Tulloch. [Feb. 2,
encircle the rectum as in Glossina. The hypopygium and penis are of the
same type as in Glossina.
x 24.
Fic, 2.—Male Generative Organs of Stomoxys. Alimentary canal dissected out to show
Malpighian tubes. Dorsal view. S.G., salivary gland; O., origin of Mal-
pighian tubes; M.T., Malpighian tubules; T.T., thickened terminations of
the tubules of one side; R., rectum; R.P., rectal papille, three of the four
are seen through the transparent rectal wall; T., testis; D., duct of testis ;
C.D., common duct ; V.S., vesicula seminalis.
Female Generative Organs.
The sex of a Stomoxys can be easily ascertained by inspection of the
hind end of the abdomen; but, unlike Glossina, the scutellar bristles are of
the same length in both sexes. The female generative organs are of the
house-fly type. There are two ovaries (fig. 3, O., and fig. 4), each consisting
1906. ] The Internal Anatomy of Stomosxys. 529
of some 60 ovarioles. The ovary is moored to the body wall by a profusely
branching trachea, which arises from the pleural space and ramifies among the
ovarioles. In the natural position the ovaries lie with the long axis of the
ovarioles pointing upwards towards the dorsal surface. Lach ovariole
contains never more than four ova in various stages of development.
The ovaries vary in size according to the degree of maturity of the lowest
ova. In some flies they occupy more than half of the whole abdominal space.
The ovarioles open into a wide tubular duct which joins its fellow from the
other ovary like the upper limbs of a Y. Asa result of this junction is formed
the common oviduet (fig. 3, C.0.), which runs down, forming a long third limb
to the Y. Below the attachment of the uterine appendages the oviduct
continues as the uterus.
The appendages consist of the uterine glands and the receptacula seminis.
The uterine glands (fig. 3, U.G.) are two rather stout tubular organs with
slightly bulbous extremities. The bulbous end is firmly joined to the lateral
oviduct by a very short double strand of connective tissue. Each gland ends
in a short fine duct, and these ducts enter separately the shallow constriction
which forms the arbitrary division between the oviduct and uterus.
The receptacula seminis (fig. 3, R.S.) are two small, black, spherical bodies,
each with a cellular socket resembling the fitting of an acorn cup. From
this runs a very fine duct which enters the division between the oviduct and
uterus in the mid-dorsal line. The receptacula are attached to each other,
but can be separated by dissection. The distal portions of the two ducts are
quite separate, but later each duct enlarges slightly, and from this point on
to its insertion is closely attached to its fellow. This portion can, however, be
separated by dissection, and it is then seen that the ducts are distinct and
enter separately.
The uterus (fig. 3, U.) is a tube of the same diameter as the common
oviduct above, and runs down in the middle line into the ovipositor. The
ovipositor (fig. 3, O.P., and fig. 4) consists of three cylindrical segments of
thin chitin, which usually lie telescoped inside the abdomen. There is also a
single external flap of dark chitin which lies folded up on the ventral surface
of the fly. When the ovipositor is extruded by squeezing the fiy’s abdomen,
the receptacula and uterus are pulled down with it, and can be seen through
the transparent walls. :
The upper segment of the ovipositor has three narrow ribs of dark chitin in
its long axis, two dorsal and one ventral. The next segment is similar. The
last segment has two dorsal plates only. The external flap, which is probably
the third rib of the last segment, is, roughly, quadrilateral, and has two
divergent prong-like processes arising from its free border.
580° The Internal: Anatomy of Stomoxys. L00e
Ar R.
Fig.. 3.
x 24.
te) % Fie. 4. Fic. 5.
Fre, 3.-—-Female Generative Organs. The natural position of the parts has been consider-
ably altered for the sake of clearness. Dorsal view. O., ovary ; R.O., right
‘ ‘oviduct ; C/O:, common oviduct ; U., uterus; O.P., ovipositor; U.G., uterine
nyuni ©: )) gland ; R.S., receptacula seminis; R., terminal position of rectum cut short
above.
Fic. 4.—Mature ovary from another specimen. F
Fig. 5.—Ovipositor extended. Dorsal view. The ventral ribs of chitin in the two upper
segments are not shown.
Specificity of the Opsonic Substances in the Blood Serum. 5381
‘The points of the chitinous ribs which strengthen the segments of the
Ovipositor project above the upper border of the segment, and to them are
attached the muscles of the ovipositor. The narrowed terminal portion of
the rectum enters the ovipositor on the dorsal surface of the uterus and runs
down to the anal opening between the external plate and the last segment.
The Specificity of the Opsonic Substances in the Blood, Serum.
By WiiiAm Buttocu, M.D., and G. T. WESTERN, M.A., M.B.
(Communicated by Leonard Hill, F.R.S. Received February 15,—Read
March 1, 1906.)
(From. the Bacteriological Laboratory, London Hospital, E.)
A relatively high degree of specificity has been demonstrated for most of
the antibodies which exist in immune sera, ¢.g.,in the case of agglutinins,
lysins, preecipitins, antitoxins. With normal sera the proof of specificity is
often difficult on account of the fact that the antibodies are present in the
majority of cases only in small quantities.
The following experiments are concerned with the specificity of the
opsonic substances of normal and immune sera. As is well known, these
opsonic substances, discovered by Wright and Douglas, act on bacteria
in such a way that the latter become an easy prey to the phagocytic
leucocytes.
Ifa given serum be tested it will be found to exert an opsonic action on
more than one kind of bacterium, and the question we have sought to
answer is whether there is one or more than one opsonic substance; in
other words, whether the opsonins are specific for the different bacteria on
which they exert their opsonic action.
In a previous communication* one of us (B.) has shown that when a
microbe, ¢.9., staphylococeus, is digested with normal serum at 37° C. for
15 minutes, and the cocci are then brought down by the aid of a centrifuge,
the supernatant liquid is found to be devoid of opsonie action for staphylo-
cocci. Where the contact of the microbe with serum has been sufficiently
long, and the centrifugalisation has been complete, the opsonin for the
particular microbe is totally removed.
* “Roy. Soc. Proc.,’ vol. 74.
532 Dr. W. Bulloch and Mr. G. T. Western. _—_[Feb. 15,
We have attempted to determine whether the opsonins are specific by
experiments of two kinds :—
1. The first method consisted in estimating the opsonic content of a given
serum towards two different bacteria. A suspension of one of these bacteria
was digested with the serum, and the mixture was thereafter centrifugalised,
the resulting supernatant liquid being tested on both kinds of bacteria. To
a quantity of the supernatant liquid the second bacterial suspension was
added, and after the lapse of a certain time the centrifuge was again applied,
and the resulting liquid was again tested.
2. The second method consisted in estimating from day to day the opsonic
content of the serum of human beings suffering from lupus. At certain
periods tubercle or staphylococcus vaccines were inoculated, and the effect
on the two opsonic curves was determined.
1. Experiment on the opsonic action of normal human serum towards
Staphylococcus aureus and Bactervum pyocyaneum respectively.
Normal human serum was mixed with an equal volume of a suspension of
Staphylococcus awreus, and the mixture was placed in the incubator for
1 hour at 37°C. At the end of this time the mixture was centrifugalised,
the supernatant liquid “A” being removed from the deposit of cocci by means
of a pipette. The supernatant liquid was in part retained, the remainder
being digested for 1 hour at 37° C. with a suspension of Bacterium
pyocyaneum, the latter being finally brought down as a deposit in the centri-
fuge, leaving a supernatant liquid “B,” which was pipetted off.
Result.
1. Normal serum (1 in 2 dilution)+staphylococci +leucocytes = 22°9) .
2 55 » (lind ,, )+2B. pyocyanewm+ 3 = 407 2.9
3. ” ” (1 in 4 a a ” at ” ool E =
4, Fluid “A” + staphylococcus + a = 05182
5 Be SOI? + B. pyocyaneum + » = +0| a x
Cae eB sts » ie ” = O04
The contact of the serum with staphylococcus leaves the opsonic action of
the serum for Bacterium pyocyaneum practically unchanged, the pyocyanic
opsonin being finally removed by contact of the serum with this microbe.
A similar result was obtained when the serum was brought to act on
staphylococcus and tubercle -bacillus, as- can be seen in the following
experiment.
1. Normal human serum was mixed with an equal quantity of an emulsion
of tubercle bacilli in 0°85 per cent. NaCl solution. The mixture was digested
for 30’ at 37° C. and then centrifuged. In this way a deposit and a super-
natant liquid “ A” was obtained.
533
of Opsonic Substances in Blood Serum.
uty O
1906.] Specific
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534 | Dr. W. Bulloch and Mr. G. T. Western.” [Feb. 15,
. Normal human serum was mixed with an equal quantity of an emulsion
of es ylococcus aureus in 0°85 per cent. NaCl solution. The mixture was
digested for 30’ at 37° C. and then centrifuged, a supernatant liquid “B”
being obtained. |
3. The fluid “A” was mixed with an equal quantity of an emulsion of
Staphylococcus awreus. The mixture was digested for 30’ at 37° C. and a
deposit separated from a fluid “C” by the centrifuge.
4. The fluid “B” was mixed with an equal quantity of an enulen of
tubercle bacilli. The mixture was digested for 30’ at 37° C., anda deposit
separated from a fluid “D” by the centrifuge. : eh
The opsonic content of the serum and of the fluids “A” «B” «€. Again, there is a form consisting of a bent figure
with symmetrical thickenings which form one aspect may be represented
thus:—Q. Another form consists of an asymmetrical annulus with unequal
sides, thus op While, lastly, we have a long and evenly thick ring,
thus:— ©. In this way it will be seen that in the first maiotic division of
Triton there are to be found six varieties of gemini, and upon looking into
the matter, we are led to conclude that all these six varieties co-exist in
every instance of the first maiotic division.
In some cases, however, it is possible to see more than one representative
of any particular type in one and the same cell, and upon counting the
maximum representation of any type found in a particular element, we find
in Triton this number is two. Taking each of the six classes or types of
gemini in turn, we find that cells may be found that show two representa-
tives of every one of the six classes.
566 Messrs. J. E. S. Moore and G. Arnold. Ewmistence [Dec. 13,
In Triton the number of the pre-maiotic chromosomes is 24. These in the
synapsis unite to form 12 gemini, and consequently we are driven to the
conclusion that in the first maiotic spindle figure there exists a pair of gemini
belonging to each of the six different types.
The fact that the varieties are constant in the early spindle figure, really
in itself precludes the possibility of the different forms having anything to do
with the fortuitous manner in which the gemini may become attached to
the spindle fibres, and this indication is enforced to the point of proof by the
further observation, which can be readily made, that all six varieties of
gemini are present in cells before the nuclear membrane has disappeared,
that is to say, before the spindle fibres have ever acted on them. In fig. 2 we
have a drawing of five cells in three of which the nuclear membrane is not
yet ruptured, but in each of these three cells representatives of the different
classes of gemini are as clearly to be discerned as they are on the early
spindle figures.
a 2) c dad e a
Um
Man
2
(6) (a)
2
won 10 G4 80
2 2 (a) 2 (bd)
a
19 /8lo) fq ay
Cockroach @ é Qi
Diagram showing the Forms of Heterolytic Gemini in various Animals.
Total number Total number
of gemini. of gemini.
Mian (i. saictescostes 16 Triton, sp. ......... 12
Rati cusscateiects 16 Cockroach ......... 16
Another feature which should be noted is the fact that we do not encounter
transitional forms passing from one form of the gemini to another; there is
1905.| of Permanent Forms among Chromosomes, etc. 567
no half-way set of gemini between the forms a and 0 given in the table on
p. 566, or between ¢ and d, ¢ and f, and so on.
The gemini in Triton, as we believe is the case in every other instance of
the first maiotic division, are produced by the conjugation of premaiotic
(somatic) chromosomes in pairs during the synaptic rest ;* and since there
are 24 premaiotic chromosomes in the particular instance of Triton, it follows
that there must be only four individual chromosomes which can unite with
each other to form the two gemini belonging to each of the six types.
The above results, based upon a study of the gemini in the first maiotic
divisions in Triton, are interesting in themselves, but they immediately raise
the further question as to whether the order here observed is simply a
curious instance, or an individual expression of a wider law; on account of
this we have studied in a similar manner the first maiotic division in man,
rats, and Periplaneta ; that is to say, in two more typical vertebrates and a
representative arthropod.
In the testes of rats the first maiotic division occurs in groups of cells,
and it is by no means difficult to bring under observation in a short time
thirty or forty instances of the early spindle and late prophase. In this we
have material amply sufficient to arrive at a decision upon the matters with
which we are concerned. Fig. 3 is a drawing of a portion of a tubule from
the testes of a piebald rat. It shows the outer wall of the tubule with some
premaiotic nuclei and three dividing cells belonging to the first maiotic
(heterotype) mitosis.
In these it will be seen that the gemini are of very different forms,
and that as in Triton the same forms are repeated in different
individual cells. Analysis of a large number of similar cells reveals the
fact that in this particular example there are again six varieties of gemini
(see table, p. 566). In the rat, however, instead of the premaiotic
chromosomes being 24 in number as in Triton, there are 32; consequently
we shall have to ascertain the relative numbers of the different types.
Further it will be seen that the six types present in the rat are not all the
same as those catalogued for Triton (table, p. 566). It will indeed be obvious
from this that only four of the Amphibian varieties are represented in the
mammal; to these four two new forms of gemini are added.
In regard to the type / in the table, p. 565, it is obvious that this particular
form of the gemini might be regarded as an opened out VU, which in this case
appears as a straight rod; but although this is so, the fact remains that the
* Farmer and Moore, ‘Roy. Soc. Proc.,’ 1903, loc. cit.; Farmer and Moore, ‘ Quart.
Journ. Micr. Sci.,’ vol. 48, doc. cit. ; Moore and Embleton, ‘ Roy. Soc. Proc., 1905, doc. cit. ;
Moore and Walker, ‘Thomson Yates Reports,’ loc. cit.
568 Messrs. J. E.S. Moore and G. Arnold. Eaxistence [Dee. 13,
types / and fare present together in rats in such a manner as to suggest that
they are really distinct entities.
In rats we have said that the number of the pre-synaptic chromosomes is
32 and the number of gemini is 16. These latter bodies are grouped into
six varieties, and consequently the number of each variety in rats must be
unequal.
If in rats in a large number of division figures the maximum number of
all the six varieties of gemini are counted, as was done in Triton, the results
are as follows: a—4, b—2, c—4, e—2, f--2, h—2.*
Upon comparing the above results with similar results in man we find
that here the varieties remain the same as in rats, but the relative numbers of
these varieties are again changed (see fig. 6, Plate 25, and table, p. 566), the
arrangement in man being as follows: a—2, b—2, c—6, e—2, f/—2, h—2,
For any one species the numbers of types of gemini, so far as we have gone,
appears to be constant, and the same types are retained in the case of fairly
remote genera, such as Homo and Mus; but in these genera the relative
numbers of the different kinds of gemini may vary with, or independently of,
the number of the premaiotic chromosomes.
Passing from the above vertebrate examples to the old arthropodean type
Periplaneta we find, as fig. 5 and the table on p. 566 will show, that here the
number of the types of gemini is reduced from six to five.
Upon consideration of the table it will be seen also that three of the
amphibian and mammalian types are retained, but no new type is added, and
two of the types common to both the other groups are altogether wanting.
In Periplaneta there are 32 premaiotic chromosomes and 16 gemini, so
that here, as in the case of man and rats, the number of similar forms must
be unequal.
Counting the maximum number of any type in a number of cells, as was
done in the former cases, we get the relative number of the five types in each
cell as follows: a—4, b-—4, f-—-2, g—4, h—2.
The possible bearing of the above observations upon the various existing
theories of hereditary transmission, and especially in relation to the Mendelian
hypothesis, will be obvious enough; but we feel a great reluctance at the
present time in any way to augment the obscuration of the facts by putting
forward crude theoretical anticipations.
What appears to us of first importance is the recognition of the actual
existence of permanent structural types in the gemini of different forms.
Secondly, it would appear that in any particular form the number of gemini
* It is an interesting and important fact that the number of premaiotic (somatic)
chromosomes is not the same in rats as in mice. In the latter the number is 24.
1905.] of Permanent Forms among Chromosomes, ete. 569
of each type have a constant numerical relationship to each other. Thirdly,
so far as the investigation has at present gone, certain types of gemini appear
to be common to all the widely sundered forms examined. Still further, it
will be seen that the number of different types of gemini is less in the oldest
evolutionary form Periplaneta.
Whether this last indication will be found to hold good is a matter upon
which it would at present be useless to speculate; but the fact itself opens
up a line of future inquiry which is certainly full of possibilities.
It seems to us, moreover, that it should be emphasised that both in regard
to the permanent types of gemini and their numerical relationships, as well
as with respect to the numerical constancy in the chromosomes themselves
and their periodical reductions, we are face to face with constant arrange-
ments in the parts of the unit of living substance (the cell) which seem to
underlie and to be quite independent of those external interactions that are
supposed to have helped to build the grosser features of living things.
With regard to the different types of gemini, it should further be pointed
out, that the existence of these types implies substantive differences between
the chromosomes that can unite to form the different kinds. It must be
remembered that each of the gemini arises through an association of optically
similar premaiotic chromosomes, but that at the time the nuclear membrane
is about to disappear these associations have assumed different forms. They
cannot do this unless they are of a different nature. The fact that there exist
in those nuclei which we have examined groups of similar gemini shows that
there must be sets of premaiotic chromosomes which in the synapsis can
conjugate with each other, but not with the remaining individuals.
The present position may be in part summed up as follows:—In the
fertilised egg the paternal and maternal chromosomes divide independently on
the spindle of the first segmentation figure. And they go on dividing in a
similarly independent manner throughout the soma, and during the pre-
maiotic history of the reproductive elements themselves. In the synapsis
which ushers in the maiotic phase the chromosomes unite in pairs, and in
those cases we have as yet examined only certain individual chromosomes
are capable of uniting with one another to form differing group of gemini;
in each of these groups the number of gemini is more than one, and it varies
in the different species hitherto observed.
Thus whether the conjugation of the chromosomes in the synapsis is
really the final consummation, after many generations long delayed, of the
copulatory intentions of the paternal and maternal elements, is a matter
upon which there is as yet no actually conclusive evidence.
570 Haxistence of Permanent Forms among Chromosomes, etc.
DESCRIPTION OF PLATES.
PLatTE 24.
Fie. 1.—Groups of cells from the testis of a Triton. They are all in phases of the first
maiotic (heterotype) division, and similar forms of gemini (heterotype chromo-
somes) are to be seen in numbers of different cells, as at b, ¢, d, g, f.
Fie. 2.—Cells from the testis of a Triton, all in the late prophase of the first maiotic
division. In three of the cells the nuclear membrane is still present ; but the
gemini have already assumed the same forms as those represented in fig. 1,
b, ¢, d, gm St
Fig. 3.—Cells from the testis of a rat, showing similar gemini in the different cells,
Oh Oy Gif [ep
PLATE 25.
Fic. 4.—Cell from testis of a rat. Harly phase of first maiotic spindle, showing pairs of
different gemini, a, B, ¢, d, e.
Fic. 5.—Group cells from the testis of Periplaneta, showing similar gemini in each of
the three different cells, a, 6, f, g, h.
Fie. 6.—Cells from the testis of man, showing similar gemini in the three celis at a, ¢, e, h.
Moore and Arnold. Roy. Soc. Proc., B. vol. 77, Plate 24.
Hiltaor
Moore and Arnold. Roy. Soc. Proc., B. vol. 77, Plate 25.
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The Action of Pituitary Extracts wpon the Kidney.
By E. A. ScHArer, F.R.S., and P. T. HERRING.
(Received May 2,—Read May 3, 1906.)
(From the Physiological Laboratory of the University of Edinburgh.)
(Abstract.)
Intravenous injections of saline extract of the infundibular part of the
pituitary body produce dilatation of kidney vessels accompanied by
increased flow of urine; 7.¢., the extract has a diuretic action.*
With the first injection this result is accompanied by the rise of blood-
pressure and contraction of systemic arteries described originally by Oliver
and Schafer,f and since confirmed by various observers.
With subsequent injections (if the first injection were not too small in
amount) administered within a certain interval of time after the first one,
the diuresis is usually attended not by a rise of blood-pressure,f but by a
fall (depressor effect).§ This fact furnishes evidence that the diuresis is
independent of the effects upon blood-pressure and leads one to suppose
that it is produced by a special constituent of the extract.
This conjecture is confirmed by the result of treating the extract with a
peptic digestive fluid or with hydrogen peroxide. These agents tend to
abolish the rise of blood-pressure which is produced by a first injection, but
leave the diuretic effect of the extract unaltered. Reducing agents and the
action of tryptic digestive fluid leave all the active constituents of the
extract apparently unaffected.
The diuretic as well as the pressor and depressor constituents of the
extract are not destroyed by boiling. They dialyse through parchment
paper. They are insoluble in absolute alcohol and ether.
Occasionally, especially with large doses of the extract, the diuretic effect
fails to show itself. This appears to be due to the kidney vessels
participating in the general vascular constriction which is caused by the
extract. More often such constriction of renal vessels is only temporary,
and gives place to dilatation with free flow of urine.
Hypodermic injections produce effects similar to those caused by intra-
* See Schafer and Magnus, ‘Physiol. Soc. Proc., p. ix, in ‘Journ. Physiol.,’ vol. 27,
1901.
+ ‘Journ. Physiol.,’ 1895, vol. 18.
t W. H. Howell, ‘ Journ. Exp. Medicine,’ vol. 3, 1898.
§ Schafer and Vincent, ‘Journ. Physiol.,’ 1899, vol. 25.
VOL. LXXVIL—B. BP tt
572 The Action of Pituitary Extracts upon the Kidney.
venous injection, but of a far less marked character and coming on only
gradually and after a long interval. Introduction of the extract into the
stomach is followed by even less noticeable effects. It is inferred that the
active constituents are not absorbed by the gastric mucous membrane with
sufficient rapidity to produce the usual symptoms.
Intravenous injections of extracts from the anterior or epithelial lobe of
the pituitary body do not produce diuresis: these extracts exhibit no physio-
logical activity.
It is concluded that the infundibular part of the gland produces an
internal secretion which passes into the blood and which, both indirectly
owing to its general action upon the vascular system and directly by its
special action on the renal vessels and renal epithelium, assists in promoting
and regulating the secretion of urine; in other words, the internal secretion
of the gland is ancillary to the renal functions.
Finally the relations of the pituitary body to the functions of the supra-
renal and thyroid glands and to the production of acromegaly are briefly
discussed.
INDEX ro VOL. LXXVII. (B)
Alcock (N. H.) The Action of Anvsthetics on Living Tissues. Part I—The Action on
Isolated Nerve, 267.
Amphibia, synapsis in (Moore and Embleton), 555.
Anesthetics, action on living tissues (Alcock), 267 ;
86.
Antedon, growth of oocyte in (Chubb), 384.
Antityphoid serum obtained from goats (Macfadyen), 548.
Armit (H. W.) and Harden (A.) The Quantitative Estimation of Small Quantities of
Nickel in Organic Substances; 420.
Amnold (G.) See Moore and Arnold.
chemistry of (Moore and Roaf),
Bacillus lactis aerogenes, chemical action on glucose and mannitol (Harden and Walpole),
399.
Bacteria, Voges and Proskauer’s reaction for certain (Harden), 424.
Barometric pressure, influence of increased, on man (Hill and Greenwood), 442.
Bashford (E. F.) and Murray (J. A.) On the Occurrence of Heterotypical Mitoses in
Cancer, 226.
Bateson (W.), Saunders (E. R.) and Punnett (R.C.) Further Experiments on Inheri-
tance in Sweet Peas and Stocks: Preliminary Account, 236.
Biometrical study of conjugation in Paramecium (Pearl), 377.
Blackman (V. H.) and Fraser (H. C. I.) On the Sexuality and Development of the
Ascocarp of Humaria granvluta Quél., 354.
Blastomeres, cell communications between (Shearer), 498.
Blood serum, specificity of opsonic substances in (Bulloch and Western), 531.
Bolton (C.) A Further Communication on the Specificity and Action iz Vitro of Gastro-
toxin, 426.
Breinl (A.) Pathological Report on the Histology of Sleeping Sickness and Trypanoso-
mniasis, ete., 233.
Bulloch (W.) and Western (G. T.) The Specificity of the Opsonic Substances in the
Blood Serum; 531.
Cancer, occurrence of heterotypical mitoses in (Bashford and Murray), 226.
Carbon assimilation in plants, mechanism of (Usher and Priestley), 369.
Castor oil plant, germination of seeds of (Green and Jackson), 69.
Cell metabolism studied in oocyte of Antedon (Chubb), 384.
Cerebral cortex, mammialian, with reference to comparative histology (Watson), 150.
Chestnut mares, offspring of thoroughbred (Weldon), 394.
Chick (H.) A Study of the Process of Nitrification with reference to the Purification of
Sewage, 241.
Chloroform, etc., physical and chemical properties of solutions (Moore and Roaf), 86.
Chromosomes of first maiotic division, permanent forms among (Moore and Arnold), 563.
574
Chubb (G. C.) The Growth of the Oocyte in Antedon: a Morphological Study in the
Cell-metabolism, 384.
Coat-colour in horses, inheritance of (Hurst), 388.
Convoluta roscoffensis, isolation of infecting organism of (Keeble and Gamble), 66.
Craw (J. A.) On the Filtration of Crystalloids and Colloids through Gelatine: with
Special Reference to the Behaviour of Hzmolysins, 311.
Crystalloids and colloids, filtration through gelatine (Craw), 311.
Echinus esculentus, effects of alkalies and acids on growth and cell division in eggs of
(Moore, Roaf and Whitley), 102 ; (Whitley), 137.
Embleton (A. L.) See Moore and Embleton.
Enzyme action, studies on (Nicloux), 454.
Ewart (A. J.) On the Nature of the Galvanotropic Irritability of Roots, 63.
Farmer (J. B.), Moore (J. E. S.), and Walker (C. E.) On the Cytology of Malignant
Growths, 336.
Fertility in Scottish sheep (Marshall), 58.
Fraser (H. C. 1.) See Blackman and Fraser.
Gamble (F. W.) See Keeble and Gamble.
Gastrotoxin, specificity and action 7m wtro of (Bolton), 426.
Gelatine, filtration of crystalloids and colloids through (Craw), 311.
Germination of seeds of Ricinus communis (Green and Jackson), 69.
Green (J. R.) and Jackson (H.) Further Observations on the Germination of the Seeds of
the Castor Oil Plant (Ricinus communis), 69.
Greenwood (M., Jun.) See Hill and Greenwood.
Hemolysins, behaviour in relation to filtration through gelatine (Craw), 311 ; —— and
phagocytosis of red blood cells (Keith), 537.
Hall (A. D.) and Miller (N. H. J.) The Effect of Plant Growth and of Manures upon the:
Retention of Bases by the Soil, 1 ; and Morison (C. G. T.) On the Function of
Silica in the Nutrition of Cereals.—Part I, 455.
Harden (A.) On Voges and Proskauer’s Reaction for certain Bacteria, 424 ; —— See
Armit and Harden ; and Walpole, (G. 8.) Chemical Action of Bacillus
lactis aerogenes on Glucose and Mannitol: Production of 2 : 3 Butyleneglycol and
Acetylmethylcarbinol, 399 ; and Young (W. J.) The Alcoholic Ferment of
Yeast-juice, 405.
Henderson (E. E.) and Starling (KE. H.) The Factors which Determine the Production of
Intraocular Fluid, 294.
Hepatomonas of kala-azar, development from Leishman-Donovan bodies (Rogers), 284.
Herring (P. T.) See Schafer and Herring.
Hill (L.) and Greenwood (M., Jun.) The Influence of Increased Barometric Pressure on
Man.—No. I, 442.
Humaria granulata, sexuality and development of ascocarp of (Blackman and Fraser), 354.
Hurst (C. C.) On the Inheritance of Coat-colour in Horses, 388.
Inheritance in Sweet Peas and Stocks, further experiments on (Bateson, Saunders, and
Punnett), 236 ; of Coat-colour in Horses (Hurst), 388.
Innervation of antagonistic muscles (Sherrington), 478.
Intraocular fluid, factors determining production of (Henderson and Starling), 294.
Jackson (Henry) See Green and Jackson.
575
Kala-azar, development of the Hepatomonas of, from Leishman-Donovan bodies (Rogers),
284,
Keeble (F.) and Gamble (F. W.) On the Isolation of the Infecting Organism
(Zoochlorella) of Convoluta roscoffensis, 66.
Keith (R. D.) On the Relationship between Hemolysis and the Phagocytosis of Red
Blood Cells, 537.
Kidney, action of pituitary extracts upon (Schafer and Herring), 571.
Kidston (R.) On the Microsporangia of the Pteridosperms, 161.
Lane-Claypon (J. E.) On the Origin and Life-History of the Interstitial Cells of the
Ovary in the Rabbit, 32; and Starling (E. H.) An Experimental Enquiry
into the Factors which determine the Growth and Activity of the Mammary
Glands, 505.
Macallum (A. B.) and Menten (M. L.) On the Distribution of Chlorides on Nerve
Cells and Fibres, 165. ,
Macfadyen (A.) Upon the Properties of an Antityphoid Serum obtained from the
Goat, 548.
Malignant growths, cytology of (Walker), 336.
Mammary glands, factors which determine growth and activity of (Lane-Claypon and
Starling), 505.
Marshall (F. H. A.) Fertility in Scottish Sheep, 58.
Menten (M. L.) See Macallum and Menten.
Miller (N. H. J.) See Hall and Miller.
Moore (B.) and Roaf (H. E.) On Certain Physical and Chemical Properties of Solutions
of Chloroform and other Anzsthetics.—(Second Communication), 86 ;
and Whitley (E.) On the Effects of Alkalies and Acids, and of Alkaline and Acid
Salts, upon Growth and Cell Division in the Fertilized Eggs of Echinus esculentus,
102.
Moore (J. E. 8.) and Arnold (G.) On the Existence of Permanent Forms among the
Chromosomes of the First Maiotic Division in Certain Animals, 563 ; and
Embleton (A. L.) On the Synapsis in Amphibia, 535 ; see also Farmer,
Moore and Walker.
Morison (C. G. T.) See Hall and Morison.
Murray (J. A.) See Bashford and Murray.
Nerve, action of anzesthetics on (Alcock), 267.
Nerve cells and fibres, distribution of chlorides in (Macallum and Menten), 165.
Nickel, estimation of small quantities in organic substances (Armit and Harden), 420.
Nicloux (M.) Studies on Enzyme Action—Lipase, 454.
Nitrification, process of, and sewage purification (Chick), 241.
Opsonie substances in blood serum (Bulloch and Western), 531.
Ovary of rabbit, interstitial cells of (Lane-Claypon), 32.
Paramecium, biometrical study of conjugation in (Pearl), 377.
Pearl (R.) A Biometrical Study of Conjugation in Paramecium, 377.
Pearson (H. H. W.) Some Observations on Welwitschia mirabilis, Hooker, f., 162.
Phagocytosis of red blood cells and hemolysis (Keith), 537 ; spontaneous, and that
obtained with heated serum of tubercle-infected patients (Wright and Reid), 211.
576
Pituitary extracts, action upon kidney (Schafer and Hegring), 571.
Plant growth and manures, effect on bases in soil (Hall and Miller), 1.
Plants, carbon assimilation in green (Usher and Priestley), 369.
Pleuronectes platessa, effect of acids, etc., on development of eggs of (Whitley), 137.
Priestley (J. H.) See Usher and Priestley.
Pteridosperms, microsporangia of (Kidston), 161.
Punnett (R. C.) See Bateson, Saunders, and Punnett.
Regeneration in polychzte worms (Watson), 332.
Reid (S. T.) See Wright and Reid.
Rivers (W. H. R.) Report on the Psychology and Sociology of the Todas and other
Indian Tribes, 239.
Roaf (H. E.) See Moore and Roaf, and Moore, Roaf and Whitley.
Rogers (L.) Further Work on the Development of the Hepatomonas of Kala-Azar and
Cachexial Fever from Leishman-Donovan Bodies, 284.
Roots, nature of galvanotropic irritability of (Ewart), 63.
Saunders (E. R.) See Bateson, Saunders and Punnett.
Schafer (E. A.) and Herring (P. T.) The Action of Pituitary Extracts upon the Kidney,
571.
Sewage purification, nitrification in relation to (Chick), 241.
Seward (A. C.) The Araucariez, Recent and Extinct, 163.
Shearer (C.) On the Existence of Cell Communications between Blastomeres, 498.
Sheep, fertility in Scottish (Marshall), 58.
Sherrington (C.S.) On Innervation of Antagonistic Muscles. Ninth Note.—Successive
Spinal Induction, 478.
Silica, function of, in nutrition of cereals (Hall and Morison), 455.
Sleeping sickness and trypanosomiasis, histology of (Breinl), 233.
Soil, effect of plant growth and manures upon retention of bases by (Hall and Miller), 1-
Spinal induction, successive (Sherrington), 478.
Starling (E. H.) See Henderson and Starling ; Lane-Claypon and Starling.
Stomoxys, internal anatomy of (Tulloch), 523.
Synapsis in amphibia (Moore and Embleton), 555.
Todas, psychology and sociology of (Rivers), 239.
Trypanosomiasis, histology of, and comparative changes in 7. Gambiense and other
trypanosome infections (Breinl), 233.
Tubercular infection tested by examination of blood and tissue fluids (Wright and Reia),.
194 ; phagocytosis obtained with serum of tubercular patients (Wright and Reid),
211.
Tulloch (F.) The Internal Anatomy of Stomoxys, 523.
Usher (F. L.) and Priestley (J. H.) A Study of the Mechanism of Carbon Assimilation
in Green Plants, 369.
Voges and Proskauer’s reaction for certain bacteria (Harden), 424.
Walker (C. E.) See Farmer, Moore, and Walker.
Walpole (G. S.) See Harden and Walpole.
Watson (A. T.) A Case of Regeneration in Polychzte Worms, 332.
577
Watson (G. A.) The Mammalian Cerebral Cortex, with Special Reference to its
Comparative Histology. I.—Order Insectivora, 150.
Weldon (W. F. R.) Note on the Offspring of Thoroughbred Chestnut Mares, 394.
Welwitschia mirabilis, observations on (Pearson), 162.
Western (G. T.) See Bulloch and Western.
Whitley (E.) A Note on the Effect of Acid, Alkali, and certain Indicators in arresting
or otherwise influencing the Development of the Eggs of Pleuronectes platessa and
Echinus esculentus, 137. See also Moore, Roaf, and Whitley.
Worms, regeneration in polychzte (Watson), 332.
Wright (A. E.) and Reid (S. T.) On the Possibility of Determining the Presence or
Absence of Tubercular Infection by the Examination of a Patient’s Blood and Tissue
Fluids, 194 ; On Spontaneous Phagocytosis, and on the Phagocytosis which
is obtained with the Heated Serum of Patients who have Responded to Tubercular
Infection, ete., 211.
Yeast-juice, alcoholic ferment of (Harden and Young), 405.
Young (W. J.) See Harden and Young.
Zoochlorella of Convoluta roscoffensis, isolation of (Keeble and Gamble), 66.
END OF THE SEVENTY-SEVENTH VOLUME (SERIES B).
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The Effect of Plant Growth and of Manures upon the Retention of Bases by
the Soil. By A. D. HALL, M.A., and N. H. J. MILLER, Ph.D. . ; I
On the Ongin and Life History of the Interstitial Cells of the Ovary in the
Rabbit. By JANET E. LANE-CLAYPON. (Plate 1) Rete s Ser tena ae
Fertility in Scottish Sheep. By FRANCIS H. A. MARSHALL, M.A.
(Cantab.), D.Sc. (Edin.), Carnegie Fellow, University of Edinburgh : 58
On the Nature of the Galvanotropic Irntability of Roots. By ALFRED J.
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Further Observations on the Germination of the Seeds of the Castor Oil
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On certain Physical and Chemical Properties of Solutions of Chloroform and
other Aneesthetics—A Contribution to the Chemistry of Anzesthesia.
(Second Communication.) By BENJAMIN MOORE, MA., D.Sc,
Johnston Professor of Bio-chemistry, University of Liverpool, and
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On the Distribution of Chlorides in Nerve Cells and Fibres. By A. B.
MACALLUM, M.A., M.B., Ph.D., Professor of Physiology, and Miss M. L.
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Further Experiments on Inheritance in Sweet Peas and Stocks: Preliminary
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The Factors which Determine the Production of Intraocular Fluid. By
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On the Filtration of Crystalloids and Colloids through Gelatine : with special
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Medical Association Research Scholar . : ‘ : ; ; sth oud
A Gee of Regeneration in Polychete Worms. By ARNOLD T. WATSON,
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On the Cytology of Malignant Growths. By J. BRETLAND FARMER,
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~On the Sexuality and Development of the Ascocarp of Humaria granulata
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A Study of the Mechanism of Carbon Assimilation in Green Plants. By
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The Growth of the Oocyte in Antedon : a Morphological Study in the Cell-
Metabolism. By GILBERT C..CHUBB, D.Sc., Assistant to the Jodrell
Professor of Zoology, University College, London. (Abstract) . 384
On the Inheritance of Coat Colour in Horses. By C. C. HURST : Re tole
Note on the Offspring of Thoroughbred Chestnit Mares. By
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A Further Communication on the Specificity and Action in Vitro of
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The Influence of Increased Barometric Pressure on Man.—No. |. By
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Studies on Enzyme Action—Lipase. By Dr. MAURICE NICLOUX . . 454
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On the Function of Silica in the Nutrition of Cereals—Part I. By
A. D. HALL and C. G. T. MORISON aa. rat Met the ASS
On Innervation of Antagonistic Muscles. Ninth Note.—Successive Spinal
Induction. By C.S. SHERRINGTON, F.R.S. . : : : a .475
On the Existence of Cell Communications between Blastomeres. By
CRESSWELL SHEARER, Trinity College, Cambridge. (Plate 18) . 498
An Experimental Enquiry into the Factors which Determine the Growth and
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| Series B. Vol. 77. No. B 521.
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The Internal Anatomy of Stomoxys. By F. TULLOCH, Lieut. R.A.M.
Garps {3 é 5 3 : i ‘ , ; ‘ : area ee
ee The Specificity of the Opsonic Substances in the Blood Serum. By
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4 On the Relationship between Hzemolysis and the Phagocytosis of Red Blood
Cells. By R. D. KEITH, M.A., M.D. . 537
Upon the Properties of an Antityphoid Serum obtained from the Goat. By
ALLAN MACFADYEN, M.D. : : 3 3 : ri sy Co)
On the Synapsis in Amphibia. By J. E. S. MOORE, A.R.CS., F.LS.,
Director of the Cancer Research Laboratories, University of Liverpool,
and Miss A. L. EMBLETON, B.Sc. (Plates 20-23) BOON a! SBS
On the Existence of Permanent Forms among the Chromosomes of the First
Maiotic Division in Certain Animals. By J. E. S. MOORE, A.R.C.S.,
F.L.S., Director of the Cancer Research Laboratories, University of
Liverpool, and GEORGE ARNOLD. (Plates 24 and 25). : oh DOS
The Action of Pituitary Extracts upon the Kidney. By E. A. SCHAFER,
Ribas, candP ie HERRING, (Abstract) 9. 62. a es) oe
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