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KMID : 0378019670100120101
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New Medical Journal
1967 Volume.10 No. 12 p.101 ~ p.111
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Experimental Studies on the Hemolytic Ability of Entamoeba Histolytica
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Abstract
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The ingestion of red blood cells by Entamaeba histolytica w -, first observed by Losch in 1373, and it has been recognized as a part of nutrient of the protozoa. But recently Shaffer et al.(1951) reported that the red blood cells of rabbits produce toxic substances which inhibit the propagation of E. histolytica. Craig (1927) foud that E. histolytica had hemolytic ability, but Shaffer et al. (1903) reported. that hemolytic ability of E. histolytica was different according to the strains.
The present- study was designed to know whether the red blood cell had a toxic effect for the propagation of E. histolytica and, on the one hand, whether the protozoa had selective ability to lyse the red blood cell.
These studies were as follows.
1. Hemolytic ability of E. histolytica to human and various animal red blood cells.
2. Relationship between the strains of E_ histolytica and the hcmolytic bacteria.
3. Effect of the red blood cell for the propagation of E. histolytica.
4. Cytolytic ability of E histolytica.
Materials and Methods:
1. Cultivation of E. histolytica
Modified diphasic medium containing 4 mi. of buffered saline was used for culture at 37C., and sub-cultured at two days interval.
2. Strains of E. histolytica.
YS 1, 5, 9 and 10 which were sampled in the laboratory were used.
3. The hemolytic ability of E. histolytica
a) R.B.0 human, ox, pig., dog, rabbit and sheep.
b) R.B.C. suspension.
Each specimen of red blood cells was collected carefully in a double oxalate bottle. It was centrifuged for 15 minutes at 3, < 00 r.p.m. to sediment the cells. The cells were then washed 4 times with normal saline. Finally, the washed red blood cells were resuspended in saline and centrifuged for 10 minutes at. 1, 200 r. p. m. The sediment was defined as a standard 100% erythrocyte concentration. A 5% suspension_ was made by adding I volume of the 100% erythrocytes to 19 volumes of saline. To minimize inclusion of white blood ce Is. sediment was withdrawn from near the bottom of the tube.
The media for t1he experiment were divided into 4 groups: bacteria group which was made by adding the 0.4 m1. supernatant fluid from the original culture media into new diphasic media; amoebae and bacteria group to which 0.5 ml. sediment from the bottom of the slant of the same original culture medium was added: metabolites group to which Seitz filtrates of all the original culture media was added,and a control group. Two-tenth milliliter of the 5% suspension of R.B.C. was added to each tube. Each group was incubated at 37C. for 14-20 hours and examined the hemoglobin content as well as the viability of the amoebae.
In order to define the hemoglobin measure, two-tenth ml. of 5% suspension of R.B.C. was added in 4 ml. distilled water to make the 100% solution of hemolysis as standard. The optical density of each soulution was measured on the spectrophotometer in 540 mu wave length to obtain the standard line of the various species of R.B.C. Comparing with the standard line, hemolytic ranges were defined as follows:
010% hemolysis negative
1025 % hemolysis moderate positive
25-100% hemolysis positive f Osmolarity of each group was determined with Osmometer (Fiske)
4. Relationship of E. histolytica and hemolytic bacteria
a) E. histolytica strains YS, 1, 9, 10.
b) Bacteria Bacillus subtilis
Staphylococcus aureus
c) Red blood cell Human R.B.C.
d) Method: Active amoebae were inoculated in fresh culture media and one loop of the bacterial colony was added to it (amoebae and bacteria group).
In. another series, the hemolytic bacteria were added only to new culture media (bacteria group). 0.2% ml. of 5% human red cells were added to above two groups and the hem_ olysis examined at 20 hours after the culture.
5. E. histolytica and the hemolytic substances which were produced from enteric bacteria and hemolytic bacteria.
Hemolytic substances were obtained by Seitz filter Procedure. Hemolysis was observed dividing into two groups; human red cells and hemolytic substances, human red cells combined with amoebae and hemo lytic substances.
6. Red cells for the propagation of E. histolytica.
YS 9 strain was used for this experiment. All the amoebae in 0.02 ml. of media were counted under the microscope, and the number per 1 nil. was calculated. 0.2 ml. of 5 % rabbit and sheep red cells, and the known number of amebae were inoculated in new modified diphasic medium, and incubated at 37C. for 2 days.
7. Gelatinolytic ability of E. histolytica.
YS 1. 9, 10 strains of E. histolytica were smeared on 15% gelatin media. As control the bacteria derived from the amoebae culture media were smeared on another gelatin media. After incubation for 24 hours at 37C., the media were placed in 4C. refrigerator for 30 minutes in order to confirm the gelatinolysis.
Results:
A. Hemolytic ability of E. histolytica.
1) The hemolytic ability was different according to the strains of E. histolytica. YS 1 strain lysed the red cells of pig and sheep and slightly the ox, but not from rabbit, dog, and human. YS 5 strain lysed the red cells from ox, but not rabbit, dog, pig, sheep and human. YS 9 strain lysed the red cells of sheep, but not from the others, and YS 10 strain lysed the red cells of dog and sheep slightly, but not from the others.
2) Metabolic products of each strain of E. histolytica could not inhibit the hemolytic action of the substances from the supernatants of the culture.
´B. Relationship of E. histolytica and hemolytic bacteria.
Bacillus subtilis hemolysed the red blood cells but showed slight hemolysis when combined with YS 1 and YS 9 strains, and did not with YS 10.
Staphylococcus aureus showed no hemolysis when combined with YS 1, 9, 10, Haemolytic substances of Bacillus subtilis, Staphylococcus aureus and Escherichia coli did not hemolyse the red blood cells when they were combined with E. histolytica.
`C. The propagation of E. histolytica and red blood cells.
Red blood cells of sheep and rabbits were used for the experiment. Two sets of cultures, to which 0.2 ml. of 5% red blood cell suspension were added or not added, were used.
In the culture which rabbit red cells were added and in control, a standard number of 5800 amo.-bae per mI. were inoculated into each of them. Control tubes gave a yield of 19,600 per ml. at 48 hours -from the original number, but in the red cell-treated tubes the propagation recovered on the 6th day -and the average number was 24,750 per ml. and the R.B.C. were hemolysed on the 4th day.
In the culture which sheep red cells were added and in control, counts of the control tubes at 48 -hours gave a yield of 19,300 per ml. from 5,100 originally seeded. A yield of 23,500 per ml. in the red ,cell-treated tubes was maximum at 4th day. Complete hemolysis of sheep R.B.C. occured on the 2nd day.
D. Gelatin liquifaction test.
In the amoebae (YS 1, 9, 10) and bacteria groups, gelatin liquifaction was positive, but in the both bacteria and control groups we--e negative.
Conclusion:
Four strains of Entanweba histolytica (YS 1, 5, 9, 10) were sampled from Korea, and the hemolytic and cytolytic action of the protozoa were studied.
1. Each strain of E. histolytica has selective ability to hemolyse the red blood cells of human, pig, dog, sheep, and rabbit.
2. E. histolytica inhibits the hemolytic action of enteric bacteria.
3. Metabolic products of E. histolytica does not interfere the hemolytic action of bacteria.
4. The normal red blood corpuscles inhibit the multiplication of E. histolytica but when the cells are hemolysed it accelerates the multiplication of the protozoa.
5 E. histolytica can liquify the gelatin.
From the above results it is concluded that the hemolytic ability of Entanzoeba histolytica is different according to the species, and inhibits the hemolytic action of enteric bacteria, but multiplication is inhibited in medium containing normal red cells.
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