Butler University Botanical Studies Butler University Botanical Studies

Volume 10 Article 16

The Cultivation of Endamoeba histolytica and the in-vitro The Cultivation of Endamoeba histolytica and the in-vitro

Chemotherapeutic Testing of Amebacides Chemotherapeutic Testing of Amebacides

Maurice E. Callender

Follow this and additional works at: https://digitalcommons.butler.edu/botanical

The Butler University Botanical Studies journal was published by the Botany Department of

Butler University, Indianapolis, Indiana, from 1929 to 1964. The scientific journal featured

original papers primarily on plant ecology, taxonomy, and microbiology.

Recommended Citation Recommended Citation Callender, Maurice E. (1952) "The Cultivation of Endamoeba histolytica and the in-vitro Chemotherapeutic Testing of Amebacides," Butler University Botanical Studies: Vol. 10 , Article 16. Retrieved from: https://digitalcommons.butler.edu/botanical/vol10/iss1/16

This Article is brought to you for free and open access by Digital Commons @ Butler University. It has been accepted for inclusion in Butler University Botanical Studies by an authorized editor of Digital Commons @ Butler University. For more information, please contact digitalscholarship@butler.edu.

Butler University Botanical Studies

(1929-1964)

Edited by

Ray C. Friesner

The Butler University Botanical Studies journal was published by the Botany Department of Butler University, Indianapolis, Indiana, from 1929 to 1964. The scientific journal featured original papers primarily on plant ecology, taxonomy, and microbiology. The papers contain valuable historical studies, especially floristic surveys that document Indiana’s vegetation in past decades. Authors were Butler faculty, current and former master’s degree students and undergraduates, and other Indiana botanists. The journal was started by Stanley Cain, noted conservation biologist, and edited through most of its years of production by Ray C. Friesner, Butler’s first botanist and founder of the department in 1919. The journal was distributed to learned societies and libraries through exchange. During the years of the journal’s publication, the Butler University Botany Department had an active program of research and student training. 201 bachelor’s degrees and 75 master’s degrees in Botany were conferred during this period. Thirty-five of these graduates went on to earn doctorates at other institutions. The Botany Department attracted many notable faculty members and students. Distinguished faculty, in addition to Cain and Friesner , included John E. Potzger, a forest ecologist and palynologist, Willard Nelson Clute, co-founder of the American Fern Society, Marion T. Hall, former director of the Morton Arboretum, C. Mervin Palmer, Rex Webster, and John Pelton. Some of the former undergraduate and master’s students who made active contributions to the fields of botany and ecology include Dwight. W. Billings, Fay Kenoyer Daily, William A. Daily, Rexford Daudenmire, Francis Hueber, Frank McCormick, Scott McCoy, Robert Petty, Potzger, Helene Starcs, and Theodore Sperry. Cain, Daubenmire, Potzger, and Billings served as Presidents of the Ecological Society of America. Requests for use of materials, especially figures and tables for use in ecology text books, from the Butler University Botanical Studies continue to be granted. For more information, visit www.butler.edu/herbarium.

129

By MAURICE E. C"'LLENDER

Endamoeba his/olytiea is the casual organism of the disease, ame­biasis. Global distribution of high incidence makes this protozoan in fection an enormous public health and 'economic problem. Cul­tural behavior and the ensuing search for effective chemotherapeutic agents has stimulated widespread interest in the parasitology and associated bacteriology of this organism.

A literature far too voluminous for presentation within the limita­tions necessary for the present paper has shown that it has not been possible to cultivate E. histolyt-ica entirely free from microbial cells or their products. Apparently the concomitant microbial flora creates the physicochemical environment required by the amoeba for its con­tinued well being. In 1949, Shaffer, Ryden, and Frye (21) published results of work which has been the closest approach to the goal of completely bacteria-f ree cultmes. Their method, representing a few simplifications of an earlier one, used a medium containing a non­multiplying single species of an unidentified streptobacillus.

An adequate review of the literature dealing with the chemo­therapy of E. histolytiea is likewise beyond the limitations of the pres­ent paper. A few of the significant features may be briefly pointed out. Amebacidal properties of a number of antibioitcs have been studied. In the case of penicillin: Knoll and Howell (15) found no effect upon trophozoites. Streptomycin in concentrations of 10,000 u/ml was found to have no ef fect upon the cysts and some tropho­zoites were also unaffected. Belamuth and Wieboldt (3). Spingarn and Edelman (24) fonnd that the addition of 1000-3000 u/ml of streptomycin prolonged the survival of cultures of E. histolytica.

THE CULTIVATION OF ENDAMOEBA HISTOLY­TICA AND THE IN-VITRO CHEMOTHERAPEU­TIC TESTING OF AMEBACIDESl

1 A portion of a thesis submitted in partial fulfillment of the requirements for the Master of Science degree in the Division of Graduate Instruction, Butler' University. This paper has been greatly abbreviated for publication. The com­plete manuscript may be obtained on loan from the Butler University Library.

primary, secondary, and . resillOsa. Butler Univ. Bot. Stud.

ONCUJSIONS

c!ongation ill white and red pine witli . 1949.

ed in 1945, the storage season ted with the fact that the lowest ould explain low growth in 1945 h in 1946 (the 3d year) for

is greater in each particular year particular secondary branch for nd in 4 out of 144 possibilities.

y particular year in secondary ranches from the top of the tree in 15.1 % of the possibilities.

econdary branches has a strong from the younger toward the

i.e. from the tip of the branch or oldest "internodes" of each

r only the uppermost 8 years of planting, the amount of e1onga­

year than during the preceding es was discernible with rainfall. es nnder study were too young ing established following trans­

tgh for all years except the first en probably helps to account for II correlation.

ent the tendency for branch G ), might in fluence the first year

y affect branches initiated after

using a combination of penicillin and streptomycin in equal unitage,

Seneca, Henderson, and Harvey (19) published that 2000 u/ml kills amoeba in the first generation. However. Faust (6) found that a combination of 10,000 u of streptomycin and 5000 u of penicillin had no ef fect upon the amoeba. The present writer has found that a combination of 1000 u of penicillin and 2000 u of streptomycin per m1. can be used routinely in the isolation of trophozoites from their mixed bacterial flora.

Anderson et a1. (I) found subtilin to be an effective amebacide in dilutions up to I :400,000. Gould and Hansen (12) found that bacitracin. Polymixin B and streptomycin hydrochloride were lethal in "approximately 1/16 the minimal apparent amebicidal concentra­tions of the separate compounds when they were added together to the open or sealed cultures."

Falsenfeld et al. (7) claim, without publishing supporting data, that E. histolytica was inhibited in vitro by aureomycin, bacitracin and neomycin. Hewitt, vVallace, and vVhite (13) have found aureo­mycin to be effective in dilutions as high as I :100,000. Fuller and Faust (9) obtained good growth of E. histol)'tica in dilutions of aureomycin ranging from 1 :300,000 to 1: I ,000.000. Ishihara and I'elsenfeld (14) found that neomycin inhibited growth in concentra­

tions of 100 units/ml. Thompson et a1. (26) found chloramphenicol to be ef fective as an amebacide in dilutions up to I :3,496, but Smith et a1. (23) found it effective only up to I :1000 dilution and attributed the result to inhibition of associated bacterial flora. McCowen, Cal­lender, and Lawlis (17) have found fumagillin, a new antibiotic, to

be effective as an amebacide.

Emetine has given variable results when used as an amebacide. dilutions varying from I :10.000 to I :1.000,000 being effective (Ful­ton et a1.. 10; Dennis, Berberian, and Hansen, S; Hewitt. VlTallace and White, 13: Laidlaw, Dobell, and Bishop, 16; St. John, 2S; Good­win, Hoare and Sharp, II). The last named workers also found cabarsone and chiniofon to be effective in dilutions of 1 :10,000.

The present paper presents results from a study of several strains of E. histolytica, their cultivation on various media: and their exposure to antibiotics and known amebacides.

130

1 of E with have Dr. I Katie this ~

to thl

Some simil, caus{ other from Thes Eoec Laba of 51 slant Vln

1 The obtai InOCll

perio medi grow cui tu ment mlcn

E taker

mariz public Butle

and streptomycin in equal unitage . (19) published that 2000 u/ml

n. However. Faust (6) found that eptomycin and 5000 u of penicillin The present writer has found that

'cillin and 2000 II of streptomycin the isolation of trophozoites from

otilin to be an effective amebacide in lid and Hansen (12) found that

tomycin hydrochloride were lethal mal apparent amebicidal concentra­when they were" added together to

ithout publishing supporting data. in vitro by aureomycin. bacitracin od White (13) have found au reo­as high as 1 :100,000. Fuller and of E. Iris/ol.rfica in dilutions of

to 1: 1,000.000. Ishihara and Tin inhibited growth in concentra­et al. (26) found chloramphenicol dilutions up to 1 :3,496, but Smith p to 1 :1000 dilution and attributed d bacterial flora. "McCowen, Cal­d fumagillin, a new antibiotic, to

ttlts when used as an amebacide, 1 :1.000,000 being effective (Ful­and Hansen, 5: Hewitt, Wallace d Bishop, 16; St. Jolm. 25; Good-

last named workers also found 've in dilutions of 1 :10,000.

ts from a study of several strains \'arious media, and their exposure

., MATERIALS AND METHODS

CULTIVATION

This phase of the investigation was an attempt to establish strains of Endamorba histolytica with various single species of bacteria and with varions mixed bacterial flora. Three strains of E. hislol'ytica have been used. These strains include the F-19, originally isolated by Dr. E. C. Faust, and NIH 200. obtained f rom Dr. C. W. Rees at the National Institute of Health. Nine species of bacteria were used in this study. All bacteria mentioned in this paper are named according to the 6th edition of Bergey's 1v[0'/'lual of DelenninaJive Bacte1·iology. Some of the bacteria were selected because they had been included in similar work by other inve~tigators. Other species were selected be­cause of their similarities to organisms in the first grOllp; and still others were selected because of their widely differing characteristics f rom those of the first and second groups. Five media were utilized. These media included the Frye-Meleney (8) modification of the Doeck-Drbohlav (4) media. the Entamoeba medium of the Di fco Laboratories," the Egg Infusion medium of Balamuth (2), the medium of Shaffer and Frye (20), and a medium consisting of nutrient agar slants o\'erlayed with a serum saline mixture. Tables IV2 through VIIP show the preparation of the various media.

The method used for experiments 1, 3. 4 and 5 was as follows: The media was inoculated with a 2 mm loop of bacterial organism obtained from a 48-hour culture grown on nutrient agar slants. The inoculated media was incubated for 24 hours at 37° C. After this period of incubation, sterile rice powder (Difco) was added and the media inoculated with 05 1111 of a 72-hour cultnre of E. histaiytira grown in the Shaf fer-Frye (20) medium. The completely inoculated cultures were examined at 72 hours by taking a portion of the sedi­ment containing rice powder and debris and examining under the microscope.

Experiment 2 di ffered in that the inoculation of E. histol'ytica was taken from the sixth culture of E. histol,)'tica-Bacilh~s cereus from ex­

, Table numbers are those 0 r the original thesis. Since their results arc sum­marized in the published portion of the thcsis, the tables are omitted frolll publication becausc of printing costs. They may be secured on loan from the Butler UnivCTsity Library. •

131

periment 1. Experiment 6 was an attempt to establish the growth of E. histol:yll:ca upon a more simple medium, nutrient agar slants with an overlay of serum saline. Table X 2 lists tlle experiments with the various bacteria, amoebae and media used in each.

CHEMOTHERAPY

Five strains of Endamoeba histolytica have been used in the chem­otherapy testing. These strains include the NRS strain obtained from Dr. J. G. Shaffer, Vanderbilt University; Luna, also obtained from Dr. Shaffer; NIH 103, obtained from Dr. Rees of the National In­stitute of Health; and the previously mentioned NIH 200 and F 19. The NRS and NI HI03 have been used widely by many investigators seeking new amebacides. The NIH 200 1S a strain highly pathog'enic for laboratory animals and is being used as the infecting organism for in-V'ivo chemotherapy studies in rabbits, guinea pigs and rats. Strain F 19 has been utilized for the bulk of the work reported in this paper. The Luna strain is the most recently isolated of these five strains of amoebae. The isolation was accomplished by Dr. Shaffer at Vanderbilt Gniversity in February 1948. This was the first strain to be isolated from a human case of amoebiasis using the clear medillm of Shaffer and Frye (20). Many parallel tests were nm, using a compound against these several strains of amoebae. No ap­preciable difference was noticed in the behavior of the different strains. All of the strains employed were in regular association with mixed bacterial flora with the exception of strains grown and tested with the Shaffer-Frye (20) medium.

Test procedure I utilized a modified Boeck-Drbohlav medium, (tables IV2 and IX'). The procedure of the test is as follows:

TEST PROCEDURE I

1. Acid drug to overlay in tubes to make final desired dilution. 2. Add inoculum of 48-hour culture of E. histolytica. 3. I ncu bate 48 hou rs at 37· C. 4. Examine sediment lor presence of amobae and subculture all negative

tubes and faintly positive tubes. S. Examine subeultures sfter 48 hours ineubation at 37· C. 6. Recorcl final results from the subcultures. (Complete absence of amobae

in subculture tube is indicative of activity).

Test procedure II used the liquid Balmuth (2) Egg-yolk Infusion medium. See table VF for preparation and composition of this medium. The procedure for this test is identical with procedure 1.

132

Test proce fusion meditm

I. Inoculate

2. Incubate· 3. Examine

4. Add nece:

5. Re-incuba 6. Examine

7. Incubate ~

8. EXJmil1e'

Test proced scribed in table

1. Place 2.0 r

2. Place druf dilution.

3. Add 2.01111 4. Add 1.0 m

S. Overlay tll

6. Examine e cultures.

7. After 48 h 8. Record ,iin:

Test procedu

I. Inoculate S 2. O\'erlay wi! 3. Examine <:<1

4. Add drug t,

5. Incubate 24 6. Examine al

7. Examine su

Test procedu often used by e;

some serious fat of the egg slope erroneous reslllt~

I. Inoculate all tubes with 72-hour culture of E. Iris/oly/ica. 2. Incubate 48 hours at 37· C. 3. Examine all tubes for presence of amoebae. 4. Add necessary amount of drug to tubes to give de~ired dilution.

5. Re-incubate 24 hours at 370

C. 6. Examine all tllbes and make necessary sub-cultures. 7. Incubate subcultures for 48 hours at 37· C. 8. Examine subeultures and report.

133

Test procedure V also made use of the Shaffer-Frye medium.

1. Place 2.0 lUI of serum, saline. Penicillin G mixture in all tubes. 2. Place drug in first tube and make proper dilution to give desired drug

dilution. 3. Add 2.0 ml of streptobacillus supernatant to each tube. 4. Add 1.0 ml of inoculum 01 E. his/oly/ica to each tllbe. 5. Overlay tubes with sterile vaseline and incubate 48 hours at 37· C. 6. Examine each tube for presence of amoebae and make necessary sl1b­

cultures. 7. After 48 hours incubation at 37· C. examine the subcultures. 8. Recorel .final results.

TEST PROCEDURE IV

TEST PROCEDURE V

TEST PIWCEDURE JII

1. lnoculate Shaffer-Frye media with E. hisfol~·/ica.

2. O"erlay with vaseline and incubate 48 hours at 37 0 C. 3. Ex~mine each tube microscopically. 4. ''-dd drug to each tube to give desired dilution. S. Incubate 24 hours at 37· C. 6. Examine all tubes and make subcultures. 7. Examine subcultures after 48 hours at 37

0

C.

Test procedure I was used because it was the general method most often used by earlier workers. The medium l1sed in this test has some serious faults. Prominent among these faults is the property of the egg slope to absorb certain chemicals. This can easily give erroneous results,

Test procedme IV used the modified Shaffer-Frye medium de­scribed in table VIIP. The details are as follows:

Test procedure III also used the liquid Balamuth Egg-yolk In­fusion medium. The details of this procedure follow.

f amobae and subculture all negati ....e

im have been used in the chem­e the :\RS strain obtained from . ity; Luna, also obtained from

Dr. Rces of the National In­mentioned 1','IH 200 and F 19. d widely by many investigators

is a strain highly pathogenic used as the infecting org~nism

rabbits, guinea pigs and rats. ulk of the work reported in this recently isolated of these five s accomplished by Dr. Shaffer ry 1948. This was the first se of amoebiasis using the clear

Many parallel tests were run, ral strains of amoebae. No ap­

the behavior of the di fferent were in regular association with ion of strains grown and tested

mpt to establish the growth of iUIl1. nutrient agar slants with lists the experiments with the

'!!d in each.

ified Doeck.Drbohlav medium, e of the test is as follows:

DUI{E I

ke final' desired dilution. f J:. hislol::li((l.

s incubation at 37" C. ultures. (Complele absence of amobae i aetivity).

Bal111uth (2) Egg-yolk Infusion T"ation and composition of this

est is identical with procedure 1.

Test procedure II was the method most widely used in this paper. This method of testing utilizes a liquid medium which is low in pro­tein and which also permits luxuriant growth of the amoebae.

Test procedure IV is the same method of testing as I and II, but utilizes an entirely different type of medium. This medium should give information as to the power of the amehacide to act directly upon the protozoan. This information is not clearly seen with the preced­ing test procedures.

Test procedures III and V, although using different media, are alike in that they test the power of the drug to destroy the organism as opposed to the power of inhibiting the growth of the organism.

In test procedmes I, II and III, the inocnlum of E. his/olytica was in the range of 10,000-12,000 organism per ml of culture. This in­oculum was obtained from 125 ml flasks containing the modified Boeck-Drbohlav media. A 72-honr culture of this was gently but well shaken and a representative sample counted in the manner described by Paulson and Morgenstern (18). Dilutions were then made to give the required number of organism per m1.

\iVhen utilizing the test procedures IV and V, smaller number of amoebae in the inoculum could be used. A number of amoebae amounting to 100 or 1,000 organisms per ml was used. This ten fold difference apparently led to no di fference in results.

In all of the test procedures, many subcultures were made. All negative tubes were subcultured as were all faintly positive tubes. A I1lUTIber of heavily positive tubes were also subcultured for control purposes. Tubes designated as faintly positive contained less than 5 organisms per field when viewed through a microscope with 10 X objective and 10 X oculars. The effective dilution is that lowest dilution which is negative after the reading of the subcultures.

OBSERVATIONS AND RESULTS

CULTIVATION

Experiment 1: The inoculated cultures were examined at 72 hours by taking a portion of the sediment containing some rice pow­der and examining it under the microscope. This examination re­vealed motile organism only in the tube containing Bacillus cereus.

134

This tub( tained thl still high!

Expel uriant wil After 11 E. his/oly

Expel' the preset

Expel' completel;

Expel' of the tub

Expel' of the am at which t

Durin~

positive Cl

slip, ringc organism bel'S for P' some use

A total report. T results obt vestigator~

strated act quine, 1 :51

,cedure, an,

Test p ()ther inve the drugs ( ide demon~

to 1,500,OC

most widely used in this paper. °d mcdium which is low in pro­

t growth of the amoebae.

ethod of testing as I and II, but medium. This medium should

le amebacidc to act directly upon not clearly seen with the preced­

ough using different media. are he drug to destroy the organism g the growth of the organism.

e inoCttlun1 of E. hislolytica was ism per ml of culture. This in­

flasks containing the modified r culture of this was gently but sample counted in the manner tern (18). Dilutions were then [ organism per 1111.

res IV and V, smaller number of used. A number of amoebae

s per 1111 was used. This ten fold ference in results.

any subCttltures were made. All s were all faintly positive tubes. were also subcultured for control intIy positive contained less than through a microscope with 10 X effective dilution is that lowest

e reading of the subcultures.

AND RESULTS

d cultures were examined at 72 iment containing some rice pow­

·croscope. This examination re­e tube containing BG-eiUus cereus.

This tube was subcultured into fresh medium. Growth was main­tained through a total of 17 subcultures at which time the cultures, still highly positive for E. histolyl'icG. were discontinued.

Experiment 2: The growth of E. histolytica continued to be lux­uriant with no apparent effects from the various bacterial mixtures. After II subcultures. with no demonstrable change in the numbers of E. histolylicG present, the cultures were discontinued.

Experiment 3: Examination of 72-hour cultures failed to detect the presence of E. hisIOl)·tica in any of the tubes.

Experiment 4: This was another repeat of experiment I, and completely negative results were again obtained.

Experiment 5: No growth of E. histolytica was detected in any of the tubes inoculated.

Experiment 6: Both media used supported the growth of each ()f the amoebae-bacterial cultures through a series of 12 sub-cultures, at which time the various series were discontinued.

During this series of tests. it was noticed that if a drop of the positive cultures was placed on a glass slide, covered with a cover­slip, ringed with vaseline or paraffin and reincubated at 37° c., the organism would survive and remain highly active and in large num­bers for periods up to an additional 52 hours. This observation is of some use for classroom demonstration.

CHEMOTHERAPY

A total of 216 in -vitro chemotherapeutic tests are included in this report. Table XF shows the complete tabulation of these tests. The results obtained using test procedure I correlated well with other in­vestigators utilizing like methods. Emetine hydrochloride demon­strated activity at I :20,000, Carbarsone oxide, 1 :100,000 and Chloro­quine. 1 :500. Subtilin was the only antibiotic tested with this pro­-cedure, and no activity could be shown in a dilt1tion of 1 :500.

Test procedure II also correlated well. with the observations of ()ther investigators. This method of testing was applied to all of tbe drugs and antibiotics reported in this paper. Emetine hydrochlor­ide demonstrated amebacidal activity in a dilution range of 1 :100,000 to 1,500,000. Carbarsone oxide has amebicidal activity in the same

135·

range as Emetine hydrochloride. Chiniofon was active in the range of 1 :1,000 to 1 :5,000 range. Carbarsone demonstrated no amebicidal activity in a dilution of 1,3,000. Chloroquine clemonstrated amebicidal activity over a wide range, I :500 to 1 :5,000; but 84% of the tests performed showed activity at 1 :1,000 or lower.

Among the' antibiotics tested, Fumagillin was by far the most active substance tested. Dilutions as high as 1 :262,144,000 completely inhibited the growth and reproduction of the amoebae. This anti­biotic is especially interesting in that it possesses no anti-bacterial spectrum. Actidione demonstrated activity of a high order with a 1 :10,000,000 dilution proving effective. Aureomycin was active at 1 :64,000 dilution, with Terramycin showing activity at 1 :32,000. Chloromycetin was active in a dilution of 1 :4,000 while Bacitracin and Subtilin failed to demonstrate activity in a dilution of 1 :500.

A modification of test procedure II by overlaying the medium with liquid vaseline was also used to test the activity of Fumagillin with identical results being obtained.

Test procedure III was used in the evaluation of Fumagillin. Here again. activity could be shown in a dilution of 1 :131.072,000.

Test procedure IV was utilized to show the ability of the com­pounds to act upon the amoebae directly. The previous test procedure results are all difficult to evaluate because of the adsorption of the drug, interference of bacteria, and the action of the drug upon the bacteria. Emetine hydrochloride demonstrated activity in a range of 1 :20,000 to 1 :100.000. Carbarsone oxide continued to show ac­tivity in a range of 1 :64,000 to 1 :200,000. Chiniofon had activity at 1 :2,000 while carbarsone was active at 1 :8.000. Chloroquine faileu to demonstrate activity at 1 :500.

Fumagillin continned to demonstrate the highest amebicidal ac­tivity of any substance yet reported, with activity at 1 :131,072,000. Actidione had actiyit)' at. 1 :20.000.000 to 1 :50,000,000. Subtilill. which had failed to demonstrate activity in the earlier test procedures. now showed activity in the diltltion of 1 :20.000,000. The activit,· of Aureomycin decreased slightly to 1 :32,000 while Terramycin dropped to 1 :4,000.

136

Test procedure Activity was prcsel 000.

A review of the sible role of bacteri

Attempts ha\'e I with certain single sion supported the ~

with this single spe

I t has also been 1I0sa, Proteus''Uulga either singly or in ( growing with Baril amoebae.

Ef forts to isolat aer'Hginosa, Proteus dis. Bacillus cirw/a

The cultivation flora and with org< nutrient agar slant~

rice powder added..

A review of the is given. Several at antibiotics have beel Endamoeba history! and antibiotics test( e'"ery method. Thl phenomena. This a demonstrates no at species of bacteria. I :400,000. Tests 1

no actively multip

CULTlV.... TlON

Efforts to isolate the amoebae with pure cultures of Pseudomonas aeruginosa, Proteus vulga.l'is, Escherichia coli, M iC'rococcus epidenni­dis, Bacillus circulans and Aerobacter aerogenes also failed.

The cultivation of Endamoeba histolytica with a mixed bacterial flora and with organism "t" has been accomplished with the use of nutrient agar slants overlaid with a serum-saline mixture and with rice powder added..

137

SUMMARY

A review of the in-vitro chemotherapy of E·ndamoeba histolytica is given. Several amebacides of clinical value and many of the newer antibiotics have been tested in various methods using five strains of Endamoeba histolytica., and the results are given.. a f all the drugs and antibiotics tested, Fumagillin appears to be the most potent in every method. The antibiotic, Subtilin, presents a most interesting phenomena. This antibiotic, when tested with a mixed bacterial flora, demonstrates no amebicidal activity. When tested with a single species of bacteria, organism "t," activity is shown in a dilution of 1 :400,000. Tests made with the Shaffer-Frye medium containing no actively multiplying bacteria, reveal activity in dilutions of

eli EMOTHERAPY

A review of the cultivation of E. histolytica is given, and the pos­sible role of bacteria in such cultivation is presented.

Attempts have been made to isolate trophozoites of E. histolytica with certain single species of bacteria. Ba.cillus cereus on one occa­sion supported the growth 0 f the amoebae so that it could be cultivated with this sing\e species. Other attempts to repeat this failed.

It has also been sbown that the addition of Pseudomonas aerugi­nosa, P?'oteus'vulgaris, Escherichia coli and Micl'ococcus epidel'1nidis, either singly or in combination, to cultures of Enda11weba histoly/ica growing with Bacillus cereus failed to 'influence the growth of the amoebae.

Test procedure V was used only in the evaluation of Fumagillin. Activity was present in the dilutions of 1 :131,072,000 to 1 :262,144,­000.

,.

magillin was hy far the most igh as 1 :262,144,000 completely n of the amoebae. This anti­t it possesses no anti-bacterial

cth'ity of a high order with a ve, Aureomycin was active at showing activity at 1 :32,000.

ion of 1 :4,000 while Bacitracin

ctivity in a dilution of 1 :500.

e II by overlaying the medium test the activity of Fumagillin

to show the ability of the com­t1y. The previous test procedure

ause of the adsorption of the

the action of the drug upon the emonstrated activity in a range ne oxide continued to show ac­.000. Chiniofon had activity at at 1 :8,000. Chloroquine failed

"niofon was active in the range ne dcmonstrated no amebicidal

o(juine demonstrated amebicidal

1 :5,000: but 84% of the tests

or lower.

trate the highest amebicidal ac­,with activity at 1 :131,072,000. ,000 to 1 :50,000,000. Subtilin.

,"ty in the earlier test procec1 nres. f 1 :20,000,000. The activity of

~2,000 \vhile Terramycin dropped

evaluation of Fumagillin. Here ilution of 1 :131,072,000.

1 :20,000,000. These last observations are in correlation with the direct microscopical examinations of Anderson, Villela, Hansen and Reed (1946). Table Xl [2 compares the results reported in this paper with the results obtained by previous investigators.

COl\CLUSIOJ\S

1. Endamoeba historytica can be isolated and grown with a pure culture of Bacillus cereus.

2. Addition of various other bacterial species failed to influence the growth of E. histolytica in association with Bacillus cere;.rs.

3. lt is possible to maintain cultures of E. histolytica growing both with a mixed bacterial flora and with a single species of bacteria upon a medium consisting of nutrient agar slants overlaid with a serum saline mixture with added rice starch.

4. Three methods of testing must be included for the complete evaluation of the compounds tested. One method should include a mixed bacterial flora to determine if the agent may be inactivated by such bacteria. Another method of testing should include no actively multiplying bacteria so that the direct effectiveness of the substance against the accompanying microbial organism may be ascertained. The third method of testing should be a method whereby the eff ec­tiveness of the substance to destroy as opposed to mere inhibition can be evaluated.

S. V·lith the utilization of the fore-mentioned methods, one sub­stance has consistently proved to be highly effective and should be thorOLlghly tested in cases of human amoebiasis. This substance is an antibiotic, Fumagillin.

ACKNOWLEDGMENT

The writer wishes to express his appreciation to Dr. Ray C. Friesner for his interest and encouragement in addition to critical reading of the manuscript.

LlTERATURE CJTED

1. ANDERSON, H. H., VILLELA, G. G. HANSEN, E. L., and REED, R. K. Some physical and biological properties of subtilin aud other antibiotics. Science, 103 :419-420. 1946.

138

2. BAJ hisl

384 3.

h.isl

4. BOE Am

S. DE! of I but) 194~

6. FAl

anti bias

7. FEL

tory the

8. FRY

in tf 9. FUL

Elld 10. FUL

Am<

1949 II. GaOl

amo 12. Gou

B al orga

13. HE'" End,

14. ISH!

lozo: IS. KNO

da·m., 16. LAlI

actic 20:2

17. McC (H-. 1951

18. P.IU

detel othel Med

19. SENI

peni( Tro{

139

2. H.~LAMUTR, \V. Improved cgg yolk infusion for cultivation of Endamoeua hisfolylica. and other intestinal protozoa. /lmer. Jour. Oin. Path., 16 :380­384. 1946.

3. A)iD 'vViEEOLDT, M. L. Action of streptomycin on EndcL1IlOeba histoly/tea. ;/1 'i)itro. JOllr. Parasito!., 32 (Sllpp!.) : 10. 1946.

4. BOECK, W. AND DROOHLAV,]. The cultivation of Enda.moeua histol},tica.. Arner. Jour. Hyg.. 5 :371-407. 1925.

5. DENNIS, E. W., BmIJERJ.\I\', D. A. AND H:\NSEN, S. The amebacidal activity of bisml1thoxy p-N -glycol-arsanilate and 7-iodo-4 (1 methylA diethylamino bl1tylamino) ql1inoline diphosphate. Allier. Jour. Trop. Med., 29 :683-689. 1949.

6. F.\ UST, E. C. A study of the bacterial associates and their susceptibility to antibiotics. Tropical Diseases Stndy Section, Panel Discussion on Amoe­biasis. 1948.

7. FELSENFELD, 0., VOLlNI, 1. F., YOUNG, V. M., AND TSJ-iIHARA, S.]. Labora­tory tests with newer antibiotics on microorganisms commonly prevalent ·in the tropics. Amer. Jour. Trop. Med., 30 :449-502. 1950.

8. FRYE, '0/. W. AND MELENEY, H. E.' Liver e.xtract as a substitute for serllm in the culture medium for Endamoeba histolYlica. Science, 89 :564-565. 1939.

9. FULLER, F. ,",V. AND FAUST, E. C. Aureomycin in the cultivation of Enda.moeba histolytica.. Science, 110 :509-510. 1949.

10. FULTON,]. D., JOYNER, L. P., KING, H., OSIJOND, J. M. AND WRIGJ-iT, M. Amoebicidal action and chemical constitlltion. Proc. Roy. Soc., 137 :339-366. 1949.

11. GOODWIN, L. G., HOARE, C. H. AND SHARP, 1. M. Chemotl)erapy of amoebiasis. Brit. Jour. Pharmacol., 3 :44-48. 1948.

12. GOULD, M. _~ND HANSEN, E. L. Synergism between bacitracin, polymyxin B and streptomycin HCl, in viho tests with Endamoeba histolytica and organism "t." Federation Proc., 9: No. 1. 1950.

]3. HEWITT, R, WALLACE, W. AND \VHlTE, E. The effect of aurcomycin on Endamoeba histolytica in'i,itra. Science, 112 :144-146. 1950.

14. ISHIHARA, S. J ..\ND FELSENFELD, O. (1950). Action of neomycin on pro­tozoa. Jour. Parasitol., 35 :33. Jour. Amer. !vIcd. Assoc., 142 :260.

15. KNOLL, E. W. ANn HOWELL, K. M .. [n 1;;t1'O effect of penicillin on En.­damocba hislal)'tica. Arch. Path., 42 :594-597. 1946.

16. LAIDLAW, P.O., DOBElL, C. AND BISHOP, A. Further cxperiments on the action of emetine in cultures of Endamoeba histol),tica. Parasitology, 20 :207. 1928.

17. MCCOWEN, M. c., C.~LLENDER, M. E. AND LAWLIS, J. F., JR. Fumagillin (H-3), a new antibiotic with amebicidal properties. Seience, 113 :202-203. 1951.

18. P.~ UlSON, M. AND MORGENSTERN, M. An accurate method for the numerieal determination of Elldalnol.'ba histolytiea in vitro and its possible use with other intestinal protozoa: suggested clinical application. Amer. Jour. Trap. Med., 12 :387-400. 1932.

19. SENECA, H., HEN,DERSON, E. AND HARVEV, M. The synergistic aetion o( penicillin and streptomycin on Endamoeba histolytica cultures. Amer. Jour. Trop. Med. 29 :37-39. 1949.

l\IEKT

SE", E. L., and REED, R. K. Some tilin and other antibiOtics. Science,

appreciation to Dr. Ray C. gement in addition to critical

rial species failed to in fInence n with Ba,cillns CCTCUS.

are 111 correlation with the clerson, Villela, Han;;en and results reported in this paper

,·estigators.

nlatcd and grown with a pure

-mentioned methods, one sub­ighly effective and should be

amoebiasis. This substance is

es of E. hisfol]llica growing ith a single species of bacteria t agar slants overlaid with a ,tarch.

be included for the complete One method should include a e agent may be inactivated by ing should include no actively effectiveness of the substance rganism may be ascertained. a methocl whereby the effec­

as opposecl to mere inhibition

20. SH.wn:R. J. G. AJ\O fRYE, \'Ii. \V. (2) Studies on the growth requirements of Elldaliloeba lrislol.r:ica. I. Maintenance of a strain of F.. Irislolytieo through o,er 100 transplants in the absence oi an actively mnltiplying bacterial i1ora. Amer. Jour. Hyg., 47 :214-221. 1948.

21. SH.AFFER, J. G., RYDE:-I, F. \;1,'. AND FRYE. "V. W. Studies on the growth requirements of Endoll/oeba hislalyliea. III. The growth and multiplication of two strains of E. /';slolyl,ca in a transparent medium without the addi· tion 0 f rice flour or other particular matter and without demonstrable bacterial growth. Amer. Jour. Hyg., 47 :3~5-350. 19~8.

22. SHAFFER, J. G., RYDEN, F. \V. i\NO FRYE, \"'. '0l. Studies on the growth requirements of Endamoeba histoJ),tieo. TV. Further observations on the cultivation of E. h,:slol.rlica and other intestinal protozoa in a clear medium without demonstrable bacterial multiplication. Some mocti [ications and simplification of the medium..~mer. Jour. Hyg.. 49:127-133. 1949.

23. SMITH. R. M.. JOSLYl\', D. A., GHl'HZlT, O. M., McLE ..,!', 1. W .. JR., PEN­KER, M. A., AKO EHLICH,]. Chloromycelin: biological studies. Jour. Bac!., 55:425-448. 1948.

24. SI"I'GARN, C. L. .... KO EOELMAN, M, H. The prolollgation of the viability of cultures of E. hislolylieo by the addition of streptomycin. Jour. Parasitol., 33:4l6-~18. 1947.

25. ST. JOHN, J. G. The effect of emetin on F.l1da'lllocbo hislolylica in culture. Amer. Jour. Hyg., 18 :414-432. 1933.

26. THOMPSON, P.H·L E., DUNN, MARY c., B.'I.Y.LES, ....'\I'IT .• AND REINERTSON, ]. W. Action of Chloramphenicol (cbloromyeetin) and other drugs against Elldal1lOeba hislaly/ico i1l ~'i/ro anc\ in experimental animals. Amer. JO\lr. Trap. Med., 30 :203-215. 1950.

140

"

L '1 c a

r

Jl

tl

Ii s f a

t1

I' t-I' c

h h f

P I r· 'j

I I /