notes on points of interest which have been studied while it was
in the press.

(i). The thick-film process. — For ordinary microscopic preparations,
I c.mm. of blood is spread out thinly over, say, 4 sq. cms. of area,
and then examined either fresh or stained — at the cost of much
time (section 18 (9)). Consequently I proposed the following method
[1903]. A quantity of blood, say i c.mm., is spread over only about
1/4 sq. cm. of area, and allowed to dry. The haemoglobin is then
washed out with water, and the residue, consisting of parasites, leukids,
micrids and the stromata of the haematids, is stained by any appro-
priate method. We can thus search i c.mm. of blood in about 1/16



65] ADDENDA 639

the time, or less, required for an ordinary preparation — but the
parasites, especially P. vivax, are apt to be overlooked unless the
observer trains himself in the work. Originally I washed out the
haemoglobin with water and then fixed and stained the residue as
usual; but later I obtained better results by washing out with eosin
solution, washing this off, and then passing over a weak solution of
methylene blue. R. Ruge, however, advocates the former procedure
[1903], and L. Rogers [1908, p. 17] prefers first to fix and stain, and
then to wash out the haemoglobin with a very dilute solution of acetic
acid — which leaves the outline of the haematids without obscuring the
view. All these methods are useful. Sediment and overstaining of
the stromata are to be avoided, and the film should not be too thick.
(2). Eniimerative methods. — Very rough ones are now in use. We
count the number of parasites in a given number of fields of an
ordinary preparation — giving an enormous possible error, because we
do not know the amount of blood in each field. Or we count the
number of parasites found near a given number of leukids, the latter
being counted by a haemocytometer. Here the total estimate is the
product of the two partial counts ; and if the errors contained in the
latter are both positive or both negative (which should occur in half
the estimations), the total error may again be enormous. Direct
counting by the haemacytometer is far from easy in the case of small
parasites, owing to the depth of the fluid examined.

Since the beginning of this year (19 10), the Advisory Committee of
the Tropical Diseases' Research Fund (Colonial Office) have granted
considerable funds for the accurate study of cases of malaria in
Liverpool, and Dr David Thomson and myself have commenced this
study by elaborating improved " enumerative methods " based on my
thick-film process. A measured quantity of undiluted blood is made
into a thick-film preparation, and the total number of parasites con-
tained in the whole of it is carefully counted. The quantity of blood
used is measured by means of a graduated pipette or capillary tube.
It is necessary that this tube should have a very fine calibre to allow
a small quantity of blood, such as i c.mm., to occupy a sufficient
length of the tube to permit of accurate measurements being made.
Thus a tube of i8o/x in diameter, and of 4 cm. in length, will
contain I'oiS c.mm. (say i c.mm.) of blood. Dr Wakelin Barratt
points out that any one can make a suitable pipette by drawing out a fine
capillary glass tube, measuring its calibre by the microscope, and
calculating the length required to contain the given volume (area of



640 NOTES [Sect.

circle = square of diameter x by o'7854, and area of ellipse = product
of the two diameters x by the same figure). We are, however, making
special tubes, measuring 1/4 c.mm. or more,

Mr M. Greenwood, Jr., of the Lister Institute, informs me that the
percentage of error is e% = 67"449 / Jn where n is the number of
bodies actually counted in the measured quantity of blood. Thus the
error=i% if we have counted 4,550 bodies, 2% for 1,138 bodies, 5%
for 182 bodies, and 10% for 46 bodies. We must therefore always
count enough bodies (parasites or leukids) to reduce the error to any
required percentage. Hence the measured quantity of blood should
be small when the parasites are numerous, and large when they are
scarce. We should perhaps allow 5% error for the measurement of
the blood, especially when very small quantities are taken. First
sample should be rejected, and fixing before staining is demanded in
order to avoid loss. The method is excellent for leukids.

(3). Some results. — Applied to a case of sleeping sickness this method
disclosed, besides other facts, a regular periodic rise and fall in the
number of trypanosomes occurring every seven to eight days — a
phenomenon apparently not previously detected {^Proceedings of the
Royal Society, June 19 10). With twenty-five cases of malaria some
of our results are, briefly, as follows : —

When fewer than about 1,000 per c.mm., the sporids are not always
numerous enough to cause marked rise of temperature (section 18 (8)).
They may remain present in very small but detectable numbers during
every day between relapses. A rise in their numbers to about 1,000 or
more is generally accompanied by a febrile relapse and followed by a
fall in haemoglobin. A fall in the number of malignant sporids may be
followed by such an abrupt and high rise that the said fall cannot be
due to the death of the parasites, but must be caused by their retire-
ment from the peripheral blood — thus proving the hypothesis of
Marchiafava and Bignami. Relapses may occur without a single
crescent being found even after daily thick-film examinations made
during twenty-nine days ; and the number of sporids counted during
such relapses cannot be explained by parthenogenic reproduction,
unless we suppose that each crescent can produce some hundreds of
spores. A rise in the sporid curve is often followed after eight to ten
days by a corresponding rise in the crescent curve, suggesting that the
latter require this period for development from the former; but some-
times no crescents at all are produced, even without quinine ; or else
the gamogenesis commences or ceases suddenly. The rise of the



6s] ADDENDA 641

crescent curve may be very rapid and continuous. The fall may be
equally rapid at first, but often alternates with short rises — suggesting
continuous death and reproduction ; but later the fall tends to be more
gradual. Quinine (even in 2 gramme daily doses in a boy), soamine
and methylene blue had no very decisive effect on the crescents
when once formed. The crescents have never numbered more than
1/8 the highest number of sporids. Of the latter (malignant),
300,000 was the largest number found per c.mm. G. C. E. Simpson,
working with us, has demonstrated parallel fluctuations of urobilin
in urine and faeces. Faradic and galvanic currents applied to the
spleen did not increase the number of crescents in the peripheral
blood, and X-rays to the spleen did not prevent a relapse. (Compare
Chapter IV.)

The method gives at the same time much more accurate diagnosis,
and is useful for obtaining the parasite rate (section 51).

(4). S. T. Darling has recently done some good enumerative work
at Panama, partly pubUshed [1909] and partly just communicated to
me by letter [1910]. He counted the number of crescents in patients
(by comparison with leucocytes), fed Anophelines on them, and then
counted the zygotes in the insects ; and estimates that the mortality
of the parasites in the stomach cavity is 97% (section 18 (i)). A.
albimana, bred in laboratory, weighed o"ooo8 grammes before feeding
and o'ooi6 grammes after a moderate blood meal. He takes the average
blood-meal to weigh about one milligramme, and from the number
of gametids counted in the blood, he estimates the number which
must have been ingested during the meal. This he compares with
the number of zygotes actually found. For example, he estimated
that one mosquito should have contained 1,632 zygotes after three
feedings : but it contained only fifty. He thinks that fully half the
ingested gametids are captured by leukids in the insect's stomach —
as I showed in 1895. In one mosquito he found 168 zygotes. He
concludes that if the gametids in a patient's blood are less than about
12 per c.mm. they will not be numerous enough to infect mosquitos.
After experiments on " a number of patients," he thinks that 30 grains
of quinine taken daily will reduce the number of crescents (section 23).
We have seen, however, that the crescent curve may fall quite irregularly,
and sometimes vary rapidly, without any quinine.

At my suggestion my brother, E. H. Ross, recently carried out
the following experiments at Port Said, in parts from which mosquitos
have been entirely banished : 344 Culex fatigans, males and females,

2 S



642 NOTES [Sect.

were liberated from bottles in houses free from mosquitos, and search
was made to ascertain what became of them. Only 54, or 16%, were
found, mostly dead, during the next day or two, after which the
remainder disappeared entirely. Only three persons were bitten after
the experiment — suggesting that the biting ratio (section 29 (3)) is
very low, say 1/50.

(5). We have found three more successful mosquito inoculations
of man, bringing the number up to 38 (section 17).

Case 36, J. Tsuzuki [1902]. — Source, benign tertian. One of
many infected Myzorh. sinensis bit healthy subject in Sapporo, Jeso,
Japan, which is free of malaria on 24th August (?) 1901. Fever and
parasites on 31st August.

Case 37. N. Jansco [1903]. — Source, malignant. A. daviger
(? Meig.) bit subject on 6th December 1907 in Hungary. One gramme
sulphate of quinine given on seventh and eighth day after inoculation.
Fever and parasites on 18th December.

Case 38. Ibid. — Same source and one A. daviger. Subject given
same dose of quinine on eighth, ninth and tenth days after inoculation
on 20th and 25th October (? year). Fever on 4th November and
parasites on the 5 th.

Jansc6 also records three interesting negative cases obtained at the
same time. Case i received i gramme of quinine daily from the
second to the fourteenth day after inoculation, and remained well
during nine months' incubation. Case 2 received 1/2 gramme every
morning on an empty stomach from ten hours before inoculation to
the thirteenth day afterwards, and remained well during one month's
observation. Case 3 received i"5 grammes quinine on fourth, fifth,
ninth, tenth, fourteenth and fifteenth days after inoculation, and had no
fever during one month's observation.

M. Glogner [1905] records two cases which he thinks were due to
inoculation during vaccination.

(6). With reference to the attitude of Anophelines, Mr Theobald
informs me that the only species known to him of which the larva
suspends itself in water like a Culicine larva is Myzomyia azriki
Patton, 1905 ; and that Myzomyia culidfades is the only one of which
the adult has an abnormal attitude So far as he knows the larvae of
all the species have no siphon.



65] ADDENDA 643

(7). Regarding the enumeration of mosquitos (section 29 (4)), I
forgot to mention a method used at Panama and elsewhere which
consists in keeping note of the number of breeding-places found from
time to time. It is, of course, a very vague method, but has the
advantage of being easily practised by the working gangs or moustiquiers.
Such records give them no additional trouble and serve for a check
upon their work.

(8). A. Eysell in the Archiv. fur Schiffs und Tropai Hygiene,
Band XIV., 19 10, argues that M. rossii is the same as the malaria
bearing mosquitos of Kinoshita and also of Schiiffner.

(9). Colonel W. G. King, the distinguished Sanitary Commissioner
of Madras and Burma, informs me that from estimates which he has
made regarding the cost of death and of sickness in India, he finds
that the death of an adult costs Rs.20; of a child Rs.7 ; while a
month's sickness of an adult costs Rs.9. and of a child Rs.4'5. As
there are quite 1,000,000 deaths from malaria every year in India, with a
corresponding amount of sickness, the total cost of the disease may be
roughly computed from these figures.

(10). Some time ago I asked Professor Karl Pearson to assist me
with the calculations regarding the diffusion of malaria given in
Chapter V of this book. He requested Mr H. Waite to undertake the
work. Unfortunately Mr Waite was not able to complete his studies until
this book was finished ; but I have now seen the proof of his paper —
which I hope will appear in Biometrika. Mr Waite proceeds on the
basis given in my report on Mauritius and in section 2 7 ; and it is very
important to note that his general conclusions are the same as those
obtained independently by me and given in section 28 ; that is to say,
that the malaria rate in a locality will fall or rise to a definite stable
figure depending upon the average proportion of Anophelines present.



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