Since Boeck, W.C. and Drbohlav, J.(1925) reported their successful cultivation of Entamoeba histolytica in Lock-Egg Serum(L.E.S.) medium, scores of investigators have devised and described their own new or improved methods to grow this parasite
in an artificial environment.
The following are the media which have been cited and employed by many workers in this field; Dobell and Laidlaw's medium(1926), Craig's media(1926, 1927), Tanabe and Chiba's medium(1928), Cleveland and Colliers medium(1920), St. John's medium(1932), Tsuchiya's s.c. medium(1934), Frye and Meleney's flask cultivation(1935), Balamuth's egg yolk medium(1946), Nelson's alcoholic extract medium(1947), Shaffer, Ryden and Frye's technique by Reeves et al.(1957), axenic cultivation by Diamond L.S.(1961) and large lot cultivation by Reeves and ward(1965) etc.
These reports described the method in full detail and some of them might be devised according to the object of studies. However, the numerous and continuous devices indicate that the satisfactory culture for growth and propagation of the amebae has not yet appeared, and the reason has been considered that component of liquid portion of the culture are mainly related.
Various integrant component, which may influence the propagation of amebae, in the liqud portion of the media have been tested; horse serum, serum of whole blood of human or rabbit, egg white, extract or infusion of liver, beef extract, yeast extract and trypticase etc.
The greater growth potential of mammalian serum from newborn, as opposed to adult, individuals has been recognized by previous worker; Howe, P.E.(1921, 1922), Gey, G.O.(1929). And pederwon, K.O.(1944, 1945) showed that the sera of the new born calf contain very littel γ-and β-globulin and the fraction represented by the α^^1-globulin area contains large amounts of mucoprotein, fetuin, different from the serum albumins and blobulins.
Recently, as examplified by the reports of Puck, T.T. et al.(1958), Holland, J.J.et al.(1959) and Hsu, T.C. et al.(1960), newborn calf serum has been widely used in tissue culture technique, because of its low toxicity for cell and less inclusion of antibody.
The author tried to improve several procedures on preparing egg slant and overlay of Modified Diphasic Medium(as utilized in the Dept. of Trop. Med. &려ㅠ. Health, Tulane Univ.: Faust and Russell, 1964), and have attempted to culture E. histolytica in this medium using the serum from 3 days old calf.
The experiment has been carried on comparpatively with other species of sera.
The results are summarized in the next pages.
MATERIALS AND METHODS
Modified Diphasic Medium Consists of two parts; a solid egg slant and a liquid overlay. Procedures for preparing this medium are briefly as follows: 1) 4 whole hen's eggs are emulsified with 50ml. of Ringer's solution, 2) dispense 3ml. amounts into screw capped pyrex test tubes(1.6cm ×15.0cm), 3) autoclave at 15 1b. for 20 minutes in order to make slant and sterilize, 4) overlay each slant with about 5ml. of buffered saline9pH 7.0), 5) add a loopful of sterile powdered rice starch just prior to use.
Stock Ringer's Solution
Nacl …………………………………………………………… 70.0 Gm.
NaCl^^2 …………………………………………………………… 3.0 Gm.
KCl …………………………………………………………… 2.5 Gm.
Distilled Water …………………………………………………………… 1000 ml.
Buffered Saline(pH 7.0)
0.85% Saline …………………………………………………………… 450.0ml.
M/15 KH^^2PO^^4 ……………………………………………………………… 19.0ml.
M/15 Na^^2HPO^^4 ……………………………………………………………… 31.0ml.
The above procedures were modified from the original method as follows:
1) Chemicals were kept in dessicator more than 5 days before use, with CaCl^^2 for dry and rotary suction pump for ventilation.
2) Fisher Junbo magnetic stirrer was employed instead of glass beads in order to emulsify the hens' eggs in Ringer's solution.
3) Stock-Rigner's solution was stored in a specially devised plastic bottle, equipped with two bended fine glass tubes through the rubber plug. The one was from the bottom to open air and the other from the upper portion of the bottle to outside via glass tube which contained KOH granules.
1) Serum was prepared from 3 day-old male calf. The serum was sterilized through Seitz filter, and stored in the deep freezer (-30。C), and was inactivated before use.
2) Rabbits, dog, adult bovine and pig serum were collected from parasite free animals, and human serum from the author. The procedures of preparation were the same as (1).
Horse serum was obtained from the National Institute of Health, Seoul.
Fetal calf serum was from Grand Island Biological Co., Grand Island, New York.
C. Penicillin G
Crystalline Penicillin G sodium(Keunwha Antibiotic Medicine Co., Seoul)
Containing 4,000,000 I.U. and buffered with sodium citrate was used.
It was stored in the dessicator, and dissolved with buffered saline 9pH 7.0) before use.
The "YS-9" strain was employed in this study.
The cysts of this strain were collected from the stool of a liver abscess patient in Cheju Island and maintained more than 5 months by subculture.
The associated bacterial flora was determined as Escherichia coli and Paracolon group.
E. Amoeba count
The method which was devised by Paulson, M.(1932) and modified by Craig, G.M.(1939) was applied for amoeba counting.
the suspension of the culture was mixed gently and thoroughly, and a small amount of overlay was aspirated with a steriled capillary pipette.
The number of amoebae in 9 large ruled squared of a haemocytometer chamber(Spencer brightline improved Neubauer) was calculated.
Multiplication of this figure by 10/9 gives the number of amebae per mm**3.
In this study, the average of 4 counts of active amebae per tube was assumed as the number of amebae per mm**3 daily count.
Moreover, 5 culture tubes were observed in every experimental group, and the data of every 24 hours were treated statistically.
Amoebae were transfered into Modified Diphasic Medium from the initial culture media, and subcultured every other day. Sediments of liquid portion in 2∼3 tubes were collected. The sampled amebae were diluted with warmed buffered saline (pH 7.0) in order to adjust the number to 10,000 per 0.3ml. The given amount was
inoculated in experimental tubes.
Prior to inoculation and at the end of experiment, the pH values of liquid phase of the experimental media were measured by means of Beckmann 72 glass electrode pH meter.
H. Expeimental infection
Five young parasite free rabbits were selected for this study. Laparatomy was performed under ether anesthesia, and approximately 200,000 trophozoites were introduced in the terminal ileum by means of a 2ml. syringe. Two animals received
Animals were sacrificed 7,112 days after the inoculation, and gross and histopathologic changes were studied.
A. Amount of Calf Serum:
The media were divided into 6 groups according to the amount of calf serum; 0,1,2,3,4 and 5 drops (1drop=1/15ml).
Serum was diluted with 5ml. of buffered saline(pH 7.0) and overlayed, and inoculated with 2000 active amebae per al. The media were incubated at 37。C for 7 days and daily counts were recorded.
The results are summarized as follow:
( ) … days of culture
1. no calf serum group.
(1) 3.9…±0.6 (2) 84.8±5.2 (3) 81.3±7.3 (4) 16.7±4.7 (5) 2.7±1.3
2. 2 drops group
(1) 5.5±0.9 (2) 91.6±4.7 (3) 156.4±14.8 (4) 48.7±47. (5) 29.5±16.8
3. 4 drops group
(1) 2.7±0.7 (2) 42.6±4.7 (3) 88.5±25.0 (4) 19.7±5.3 (5) 35.2±20.5
The maximum growth was observed in 2 drops group on the 3rd day.
B. Propagation Test:
Sera were classified into 7 groups; calf, adult bovine, pig, dog, rabbit, hoser and human. 2 drops(=2/15ml.) of respective serum were diluted with 5cc. of buffered saline (pH 7.0), then overlayed, inoculated, incubated and counted.
Data of each group are as follows:
( ) … days of culture
1. calf serum
(2) 100.8±15.6 (3) 200.9±13.8 (4) 11.9±+3.2
2. adult bovine serum
(2) 66.4±7.8 (3) 94.9±10.0 (4) 14.9±5.7
3. pig serum
(2) 41.9±9.7 (3) 105.1±23.8 (4) 13.9±4.8
4. dog serum
(2) 25.9±10.9 (3) 51.7±15.3 (4) 8.7±5.5
5. rabbit serum
(2) 57.6±8.3 (3) 68.6±30.6 (4) 16.7±6.5
6. horse serum
(2) 135.4±11.1 (3) 54.9±5.7 (4) 2.4±0.8
7. human serum
(2) 127.0±21.6 (3) 34.9±17.2 (4) 4.2±1.5
The maximum growth curve was observed in calf serum group on the 3rd day. The next were horse and human serum groups on the 2nd day.
C. Calf Serum and Penicillin G:
Media were divided into 3 groups according to the amount of calf serum; 0.2, and 4 drops (1 drop=1/15ml). In each group the penicillin G was added in 3 different units, i.e. 0, 1,600 I.U. and 2,600 I.U.
As the peak growth of amebae in each group was observed to be equal on the 3rd day, the results of the data are summarized as follows;
1. no serum group
0.1U. 52.6±4.7, 1,600 I.U.48.8±10.0 2,600 I.U.60.6±0.6
2. 2 drops of calf serum group
0.1I.U. 109.3±13.0, 1,600I,U, 129.1±6.2 2,600I,U, 75.7±15.9
3. 4 drops of calf serum group
0.1I.U. 59.4±8.2, 1,600I.U. 70.1±10.5, 2,600I.U. 37.1±13.5
The maximum propagation of amebae was observed in 2 drops of calf serum group to which was added 1,600I.U. of Penicllin G.
Additionally, propagation of E. histolytica in bovine and other sera to which were added 1,600I.U. of Penicillin G were studied. The sera used were foetus calf, calf and adult bovine, rabbit, horse and human serum. 2 drops(=2/15ml.) of each serum were diluted with 5ml. of buffered saline(pH 7.0) and overlayed on the egg slant of the medium.
The peak of the growth curves of amebae in all groups showed on the 3rd and 4th day..
1. numbers of amebae on the 3rd day
fetus calf 6.7±1.4, calf 43.4±5.4,
adult bovine 1.3±0.4, rabbit 6.8±1.7,
horse 14.0±4.6 human 4.7±1.6
7. numbers of amebae on the 4th day
fetus calf 16.6±7.7, calf 13.9±7.9,
adult bovine 0.1±0.07, rabbit 3.8±1.2,
horse 13.7±3.4 human 3.9±1.5
The highest peak was observed in calf serum diluted group to which was added
1,600I.U. of Penicillin G on the 3rd day.
E. Experimental Infection of Rabbits:
Rabbits No.3(♀) and No.4(♂, control) were sacrificed on the 7th day, and No.1(♂), No.2(♂) and No.5(♀, control) on the 12th day after inoculation.
Body weights of animals were equally downed from 1.9kg to 1.6kg.
By necropsy the following numbers of gross lesions were found in the caecum.
They were covered with cream colored to yellowish exsudate, but no lesion was found in control animals,
No.1…………………1(diameter measured 4mm)
No.2…………………5(7mm…1, 3mm…1, 0.5mm…3)
No.3…………………3(15mm1, 10mm…1, 0.5mm…1)
In the histological study(rabbit Nos.2,3), flask shaped and bottle shaped ulcers were striking and the submucosa was considerably thickend.
Ragged and overhanging mucosa formed the edges of the lesions and typical necrotic processes which prevail in an amoebic lesions were observed. Trophozoites of E. histolytica were seen all lesions and were situated principally at the necrotic material near the more normal mucosal and submucaosal tissues.
Calf and rabbit, dog, pig, human, cow sera were subjected to study whether it promotes the propagation of Entamoeba histolytica in the Modified Diphasic Medium.
1) The propagation of amebae was most luxuriant on the 3rd day of culture in the group to which 2 drops9=2/15ml) of calf serum were added.
2) when 1,600I.U. of Penicillin G was added in the calf serum medium, more abundant growth of amebae occured than in other units of Penicillin G added groups.
3) The amoebae which grew in the calf serum added media produced the typical amoebic lesions in the coecum of rabbits, 7 and 12days after the exprimental inoculation.
It was concluded that Calf Serum was excellent for the culture of E. histolytic which provides richer harvest of the parasite with the less serum.