survival of ancylostoma caninum in vitro (i)1)
TRANSCRIPT
Jap. J. M. Sc. & Biol., 9, 283-292, 1956
SURVIVAL OF ANCYLOSTOMA CANINUM IN VITRO (I)1)
YOSHITAKA KOMIYA,2) KAZUO YASURAOKA3) AND ATSUSHIGE SATO4)
Department of Parasitology, National Institute of Health, Tokyo5)
(Received: October 6th, 1956)
For studies of physiology of helminths, their maintenance in vitro should be
necessary. The survival technics of several species of helminths have been
studied for relatively long periods. No such work has ever been available for
hookworm. Here the results of experiments on survival of Ancylostoma caninum ,
recently made by us are introduced.
MATERIALS AND METHODS
Ancylostoma caninum was applied for this purpose because of its facility of
obtaining. Several dogs were infected with 100 specimens of its larvae per cutan
respectively, and 3 to 4 weeks after infection each dog was dissected and worms were
removed from its small intestine safely by the use of dissecting needles . For external
Fig. 1. The inverted microscope
sterilization, the worms recovered were washed in 40 cc Petri dishes in 5 changes of sterile physiological saline solutions incorporated with antibiotics in a high concentra-tion. Then three couples of worms were transferred to each Carrel flask (3 .5 cm diam.) containing 4.8 cc of each experimental medium, to which were added previously Peni-
1) This work was supported by a Grant in Aid for Fundamental Scientific Research
from the Ministry of Education.2)小 宮義孝 ・3)安羅 岡一 男 ・4)佐藤温 重
,5)国 立予防衛生研究所寄生虫部
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cillin G potassium 50 units, Dehydro streptomycin sulfate 100ƒÊg, and 50ƒÊg of
dehydroacetic acid per 1 cc medium, and three flasks were used for each medium.
The medium was changed completely every 2 days. As the criterion of survival,
motility of worms were tested at intervals of 12 hours under the inverted dissecting
microscope of low power (vide, Fig. 1). Serum and blood cells used here were obtained
from the carotid of a hookworm free dog with an injector. All experiments were
conducted under aseptic condition.
RESULTS
1. Survival Time in Various Physiological Saline Solutions
For the survival of Schistosoma japonicum, the most efficient medium was
already proved to be Ringer's solution among various physiological saline solutions
Fig. 2. Comparison of average length of life of Ancylostoma caninum
in various physiological saline solutions (28•Ž)
(Ito and Komiya, 1955). However, here Ringer's, Locke's, Tyrode's (without
glucose), Krebs-Ringer's phosphate and bicarbonate solutions were tested as a
basal medium. Krebs-Ringer's solution was prepared using the procedure out-
lined by Umbreit et al. (1951). All Carrel flasks used were incubated at 28•Ž.
The survival time of worms in respective solutions is shown in Fig. 2,
which demonstrated Krebs-Ringer's bicarbonate solution was most efficient for
their ;survival. The male survived in this medium for 6.6 days (4.5-10.5), and
the female 7.3 days (4.5-11.5).
2. Survival Time in Krebs-Ringer's Bicarbonate Solution Added
with Glucose
Here 0.01, 0.1, 0.5, and 1.0% glucose were added respectively to Krebs-
Ringer's bicarbonate solution in which worms were kept at 28•Ž.
The survival time of worms in the medium with different percentages of
glucose was shown in Fig. 3. As seen in this figure, addition of glucose to the
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medium showed a markedly increased survival time. It is to be noticed that the
female survived longer than the male along with the increase of percentage of
glucose. in the medium with 0.5% glucose, the female survived for 21.5 days, whereas the male for 10.5 days at the maximum.
3. Survival Time in Dog Serum Diluted with Krebs-Ringer's
Bicarbonate Solution
Fig. 3. Comparison of average length of life of Ancylostoma caninum
in Krebs-Ringer's bicarbonate solution using various concentrations
of glucose (28•Ž)
Fig. 4. Average length of life of Ancylostoma canirum in various
dilution of dog serum with Krebs-Ringer's bicarbonate solution (28•Ž)
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Hoeppli et al. , (1938) stated that the serum diluted with an equal amount of
Tyrode's solution gave the best result in the case of Clonorchis sinensis. The
same result was described on Paragonimus westermani by Yokogawa et al.
(1955), whereas the survival period of Schistosoma japonicwm became longer
as the serum was more concentrated (Ito et al., 1955).
In the present experiment, worms were kept in dog serum diluted with
Krebs-Ringer's bicarbonate solution in different percentages and incubated at
28•Ž (Fig. 4). As seen in the figure, the male worms survived for 3 weeks in
50%, 75% and whole serum, and the female for 5 weeks in average. The per-
centage of serum, 50%, 75% or whole serum, seemed to be of no erect on the
duration of their survival time. In the following experiment, 75% serum was
used as the basal medium.
4. Survival Time, Copulation, and Egg Laying in Dog Serum with
and without Blood Cells
Blood cells suspension was prepared as follows : To eliminate serum as
completely as possible, 1 cc of heparinized plasma was washed with Krebs-Ringer's
bicarbonate solution by centrifugation for three times. One cc of Krebs-Ringer's
bicarbonate solution was added then to the sediment. Serum diluted to 75%
Fig. 5. Comparison of the length of life of Ancylostoma caninum in
dog serum with and without blood cells.
with Krebs-Ringer's bicarbonate solution was used either with or without the
addition of a small amount of the blood cell suspension.
Survival time : Fig. 5 demonstrates the survival of worms in the serum
with and without blood cells at 28•Ž and 37•Ž respectively. As seen in the figure,
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Table 1. Egg laying of Ancylostoma caninum living in
dog serum with, and without blood cells (37•Ž)
A...serum with blood cells.B....serum only.* .....No. of eggs including normal ones.
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the addition of blood cells to the serum had no effect on survival time. Survival
time of worms at the temperature of 37•Ž was longer than that of 28•Ž. In the
serum at 37•Ž the longest time of survival was 6 weeks in the male and 12
weeks in the female.
Copulation : During the first 3 weeks of survival time, copulation in vitro
was observed frequently. The addition of blood cells to the serum had no effect on
their copulation.
Egg laying : In the above experiment, the medium was removed every 2 days
by transfer of worms, and everytime egg laid in the medium was examined.
In the medium incubated at 28•Ž, egg laying was observed for a few day, but
at 37•Ž for a longer period (vide, Table 1). The laid eggs included some
abnormal eggs which were assumed to be unfertilized eggs (vide, Fig. 6). These
abnormal eggs were found from the initial day until the final of survival time.
In the medium without blood cells at 37•Ž egg laying was observed through the
82 day period of servival. During 3 weeks from the initial day, numerous
embryonated eggs and hatched larvae were found in the medium. After 4 weeks
they disappeared completely, but a small number of them were found later again
between the 6-7.5 week. In this experiment the addition of blood cells to the
medium had no effect.
DISCUSSION
In comparing the media used by several investigators for keeping helmenths
alive in vitro the best result was seemed to be obtained when the medium applied
was the similar substance to that of the habitat of the respective parasites.
In the case of intestinal worms, various physiological saline solutions such as
Locke's, Kronecker's and Tyrode's solution with a small amount of peptone water,
gave good results. Though hookworm is an intestinal parasite, its foodstuff is
considered mainly to be blood. Throughout the present work on maintaining A.
caninum in vitro, it is reasonable that the medium with dog serum at 37•Ž
was efficient for the survival of worms. The present method may adequately
be of use for investigation of their physiology, metabolism and reaction to harmful
agents in vitro.
Here the addition of blood cells to the serum had no better effect on their
survival, copulation and egg laying. However, to determine the role of blood
cells upon their nutrition decisively, further precise experiments should be
required in future.
SUMMARY
1. Among various physiological saline solutions, Krebs-Ringer's bicarbonate
solution was most efficient for the survival of A. caninum worms. Addition of
glucose to the solution resulted in a marked increase in their survival time.2. The survival time of worms maintained in whole serum did not differ
significantly from that of worms maintained in 50% or 75% diluted serum.
1956 ANCYLOSTOMA CANINUM 289
3. In dog serum at 37•Ž the longest time of survival was 6 weeks in the
male and 12 weeks in the female. Addition of blood cells to the serum gave no
effect on survival time.
4. Copulation in vitro took place in dog serum at 37•Ž , and fertilized
eggs were found until the 54 day.
REFERENCES
Hoeppli, R., Feng, L. C. and Chu, H. J. (1938) : Attempt to culture helminths of vertebrates in artificial media. Chinese M. J., Suppi . 2, 343-374.
Ito, J., Yasuraoka, K. and Komiya, Y. (1955) : Studies on the survival of Schistosoma
japonicum in vitro. 1. Survival in blood or serum media. Jap. J. Parasitol ., 4,12-18 (text in Japanese with English summary) .
Ito, J. and Komiya, Y. (1955) : Studies on the survival of Schistosoma japonicum in
vitro. 2. Survival in chemical defined artificial medium . Jap. J. Parasitol., 4. 258-261 (text in Japanese with English summary) .
Umbreit, W. W., Burns, R . H. and Stauffer, J. F. (1951) : Manometric techniques and tissue metabolism, 6th Ed. Burgess pub . Co., Minneapolis.
Yokogawa, M., Oshima, T. and Kihata , M. (1955) : Studies to maintain excysted metacercariae of Paragonimus westermani in vitro . Jap. J. Parasitol., 4, 388-399
(text in Japanese with English summary).
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Explanation of Fig. 6
a. Eggs laid between 1-2 day after the maintenance of the worm in vitro.
b. Eggs laid between 3-4 day. Some eggs have hatched already.
c. Eggs laid between 5-6 day. Some eggs have hatched already.
d. Eggs laid between 17-18 day. Some eggs have hatched already.
e. Eggs laid between 25-26 day. Some eggs have hatched already.
f. Eggs laid between 27-28 day.
g. Eggs laid between 43-44 day.
h. Same as g., another field.
i. Eggs laid between 47-48 day. Some eggs have hatched already.
j. Eggs laid between 49-50 day.
k. Eggs laid between 69-70 day.
1. Eggs laid between 79-80 day.
1956 ANCYLOSTOMA CANINUM
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