survival of ancylostoma caninum in vitro (i)1)

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

283

284 KOMIYA et al Vol. 9

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

1956 ANCYLOSTOMA CANINUM 285

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)


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,


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.


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.


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).

290 KOMIYA et alVol. 9

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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survival of ancylostoma caninum in vitro (i)1)

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