the relationship of soil protozoa to tuber- nature and

THE RELATIONSHIP OF SOIL PROTOZOA TO TUBER-CLE BACILLI1. 2

CHESTER RHINESNew Jersey Agricultural Experiment Station, New Brunswick, N. J.

Received for publication, September 22, 1934

The aim of the work outlined in this paper was to determinewhat r6le protozoa play in the destruction of tubercle bacilli innature and itwas carried out simultaneously with studies on thedeath rate of tubercle bacilli in soil and in association with vari-ous bacteria and fungi.

ISOLATING SOIL CILIATES

Preliminary studies on the problem dealing with the relationof protozoa to acid-fast bacteria, especially the tubercle bacilli,were largely concentrated on the relationship of a certain ciliateto several pure cultures of bacteria. This protozoan was foundto be present in great abundance in a soil taken from a plot ofthe New Jersey Agricultural College and Experiment Station.The organism was found to develop readily on many bacterio-logical media tested. It has been identified as Colpoda Steinii.As the relationship could not be determined with certainty in

mixed cultures similar to the mixed population in which protozoanaturally live in soil, a method of purifying the cultures wasundertaken. The procedure of repeated picking and washing theciliate was similar to that employed by Peters (1921). A singleprotozoan was picked with a sterile micropipette. This wasaccomplished readily under a dissecting microscope. The proto-zoan was placed in sterile medium in a depression slide. After

1 Journal Series Paper, New Jersey Agricultural Experiment Station, Depart-ment of Soil Chemistry and Bacteriology.

2 This work was carried out under the auspices of the Committee on theMicrobiology of the Soil of the National Research Council.

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about five minutes the operation was repeated and thus con-tinued through seven sterile droplets. The protozoan was finallyplaced in the desired pure culture of bacteria and allowed tomultiply. Control protozoa inoculated into nutrient broth andtested for purity were found to be free from bacteria. In theabove manner the protozoan was successfully introduced intoseveral pure cultures of bacteria in which it thrived. One of thebacteria was an unidentified small Gram-positive bacillus foundto be predominating in a mixed infusion in which the protozoangrew excellently. The protozoan was later transferred to a cul-ture of Bacillus megatherium, in which it grew profusely. Itfailed to thrive at a temperature of about 280 to 310C. in anA. aerogenes culture although on some occasions the introducedcells multiplied readily. When the temperature was lowered toabout 230 to 250C., the protozoan was found to thrive with A.aerogenes. During the summer months placing the test tubes ina pan of water in a draft of air caused a lowering of temperaturefavorable to this ciliate.

There is no reasonable doubt as to the purity of the bacterialcultures in which the protozoan was induced to grow. Controltransfers of the protozoa through sterile media constantly provedthem to be free from bacteria, because of the failure to developbacterial growth in nutrient broth or on nutrient agar. Theprotozoa could thus be freed of all the pure bacterial cultures inwhich they were grown. These bacteria would have quickly pro-duced turbidity in nutrient broth. As an additional precautionto assure purity, the protozoan was transferred twice from eachpure culture to another pure culture of the same bacterium, usingthe identical procedure employed in the original purification ofthe cultures. Culture studies did not show the presence of con-taminating bacteria.Three cells of protozoa freed from bacteria by transferring with

a pipette through a sterile media were placed in each of threenutrient broth suspensions of Timothy 1589, a saprophytic acid-fast organism. The suspensions were observed for a period ofone week. One and two days after the transfer many protozoa

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SOIL PROTOZOA AND TUBERCLE BACILLI

were found to be present. Multiplication of the protozoan hadactually occurred. The numbers were, however, rapidly reducedand after the seventh day the protozoa were no longer observedand could not be revived by transferring to fresh suspensions.Similar experiments with Avian 531 suspensions revealed thatmultiplication also occurred but even less readily than with theTimothy 1589 suspensions and no protozoa remained after threeor four days. Bacteriological studies proved that no contain a-tion had occurred during the manipulation. These experimentswere conducted at summer room temperature which was laterfound to be too high for the best growth of the protozoan. Bettersuccess was obtained with the growth of the ciliate in Timothy1589 and Avian 531 suspensions later, when a more convenientmethod of transferring the protozoan had been devised.

Because of the very tedious task of obtaining a sterile proto-zoan by the pipetting method and because only one cell could beintroduced into a bacterial culture at a time, a more satisfactorymethod was sought. The protozoan encysted upon drying andthe cysts possessed considerable resistance to heat and chemicals.The active protozoan in a liquid culture was destroyed by atemperature of 60C. in less than five minutes. A dried cultureresisted 900C. for about twenty minutes and 850C. for over twohours. Aerobacter aerogenes was found to be rather easily de-stroyed after drying. The cultivation of the protozoan in a cul-ture of A. aerogenes had already been accomplished. This pureculture of the protozoan and the bacterium was incubated at230 to 250C. One drop of a four-day culture swarming withprotozoa was added to a series of sterile test tubes. The moisturewas allowed to evaporate in an incubator kept at 370C. Thedry tubes were heated in an oven to determine the temperatureand time necessary to destroy the bacteria without destroyingthe protozoan cysts.The medium used was found to alter greatly the thermal death

point of the dry A. aerogenes. The bacteria in the drop of mediumdesignated B resisted 950C. for one hour, while the bacteria inthe dried drop from another medium, A, were almost always

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destroyed by a temperature of 70'C. in one hour. The formulaefor these media are:

"A" mediumSoil extract............................................... 100.0 cc.Peptone.................................................. 0.25 gm.Water.................................................... 900.0 cc.

"B" mediumSoil extract............................................... 400.0 cc.Peptone.................................................. 1.0 gmn.Water.................................................... 600.0 cc.

Soil extractSoil................ 1000.0 gm.Water.................................................... 1000.0 cc.

Boil ten minutes and ifiter

Bacteria become most numerous in the richer B medium. Thedifference in the thermal death point is probably influenced bythe number of cells.A dried drop of the four-day culture in the A medium heated

at 70'C. for one hour proved to be usually bacteria free, while theprotozoa remained viable. Cultures of the protozoan could beconveniently obtained by merely adding a pure culture of a bac-terium to a test tube containing bacteria-free cysts. To deter-mine the success of the method, pure cultures of the tiny Gram-positive bacillus were introduced into the test tubes containingthe protozoan cysts. Active protozoa could be observed afterone day. After several days, the protozoa swarmed through themedia. Transfers from each tube to a d-glucose broth fermenta-tion tube were then made. When A. aerogenes survived, gasformation was readily detected. The Gram-positive bacillusfailed to produce gas in the d-glucose broth. Tests were devel-oped for the detection of A. aerogenes in the presence of otherbacteria and were used to check all the transfers. The organism,A. aerogenes, was not destroyed in about 15 per cent of the heatedtubes.When nutrient broth was introduced into a test tube contain-

ing the protozoan cysts, excystation was brought about. Activeprotozoa could be noted in one or two days. After about the


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third day, no active protozoa were present. Bacteria-free brothfailed to support growth of the protozoa.

Nutrient broth suspensions of Timothy 1589, Avian 531 andan acid-fast soil bacillus were placed in the test tubes containingthe bacteria-free protozoan cysts. In the Timothy 1589 andAvian 531 cultures slow development of the ciliate occurred. Thecultures were continued by making transfers to fresh suspensionsof the bacteria. The ciliate failed to grow in suspensions of anacid-fast bacillus from soil; a few ciliates were still present on thefifth day; thereafter none were observed. No contamination ofcultures occurred to detract from the accuracy of the observa-tions. After two months cultivation, three cultures of the proto-zoan with the Avian 531 organism and three cultures of the proto-zoan with the Timothy 1589 organisms were still flourishing.Transfers to fresh suspensions were made weekly. Apparentlythe cultures could be maintained in this manner indefinitely.Colpoda Steinii has now been cultured with Avian 531 for morethan one year. No foreign bacteria have entered the cultures.Transfers of the Avian 531 culture to nutrient broth did notbecome turbid nor did the protozoan live in this clear broth. Ifa contaminating organism capable of supporting the ciliate hadbeen present, the ciliate would have continued to exist in thenutrient broth. No development of colonies occurred when theAvian 531 culture was plated on nutrient agar.

In later tests, Colpoda Steinii failed to develop in suspensionsof M. ranae, a frog tubercle bacillus and in suspensions of elevenacid-fast bacteria of soil origin. One soil acid-fast bacillus fur-nished luxuriant growth of the protozoan. The H 37 humantubercle bacillus and the Ravenel bovine tubercle bacillus failedto support C. Steinii.

C. Steinii did not grow in the filtrate from a six weeks glycerolbroth culture of Avian 531. The soluble constituents of the cul-ture did not alone fulfill the food requirements of the protozoan.

CULTURAL RELATIONSHIPS OF ACID-FAST BACTERIA AND CILIATES

This group of experiments was devised for the purpose ofdetermining the effect of protozoa upon acid-fast bacteria and

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acid-fast bacteria upon protozoa. The main possibilities con-sidered are:

1. The protozoa may be parasitized by the acid-fast bacteriaand serve as carriers of disease, as many insects serve as carriersfor mammalian parasites.

2. The acid-fast bacteria may be toxic to the protozoa andcause abnormalities in their functions and structures or retardnormal development.

3. The protozoa may utilize the bacteria as food and ingest anddestroy them.A technic was devised for staining ciliates by the acid-fast

method in order to determine whether ingestion of Mycobacteriaoccurred. The fixative found to be most successful was of theformula:

Picric acid............ 1.0 gmn.Alcohol..................................................... 200.0 cc.Formalin............ 40.0 cc.Glacial acetic acid............ 15.0 cc.

Equal parts of fixative and culture are mixed and allowed tostand at 370C. for thirty minutes. Excess fluid is removed bycentrifugation and 0.05 per cent egg albumin added. The excessfluid is again removed and the material spread on a glass slide,allowed to dry slowly and stained by the Ziehl-Nielsen method.Pure cultures consisting of C. Steinii and Avian 531 together

and likewise C. Steinji with Timothy 1589 were thus stained.Many acid-fst bacteria could be seen within the protozoa. Thenumber contained in some single protozoa was estimated at about100. Large clumps of acid-fast material were dispersed through-out the ciliate and many intact bacilli were noted. Undoubtedlyingestion had occurred.The problem was attacked upon the assumption that if multi-

plications of the acid-fast bacteria occurred within ciliates, theacid-fast bacteria would persist through repeated transfers of theprotozoa to a fresh medium containing no acid-fast bacteria.Timothy 1589 is able to support the protozoa indefinitely whenfresh transfers to nutrient broth are repeatedly made at inter-vals of about one week if surface growth of the bacterium occurs.

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SOIL PROTOZOA AND TUBERCLE BACILLI 375

As the bacteria then develop outside of the protozoa this failsto prove anything. Avian 531 fails to grow readily at the tem-perature, about 230C., favorable to the protozoa. A frog tuber-cle bacillus was therefore procured from the Henry Phipps Insti-tute at Philadelphia and used in the experiments. Six mixedcultures consisting of known pure cultures were prepared. Allcontained a tiny Gram-positive soil bacillus and in addition:

1. C. Steinii and Avian 5812. C. Steinii and Timothy 15893. C. Steinii and M. ranae4. C. cuculus and Avian 5815. C. cuculus and Timothy 15896. C. cuculus and M. ranae

The tiny Gram-positive bacillus served excellently to supportgrowth of the protozoa. The medium with which the cultureswere carried was such that the acid-fast bacteria were favored bythe presence of ammonia, phosphate and glycerol. Nutrientbroth furnished nutrients for sparse growth of the tiny Gram-positive bacillus which could not use the glycerol. All the myco-bacteria were capable of growing on the surface when carefullyplanted, even with the Gram-positive bacteria contaminating theliquid.

Medium "D"Nutrient broth............. 50.0 cc.Water..................................................... 950.0 cc.Glycerol................................................... 0o.1cc.Ammonium sulfate............. 1.0 gm.Phosphate buffer..............10.0 cc.

Phosphate BufferK2HPO4................................................... 60.0 gin.KH2PO4................................................... 20.0 gm.Water..............1000.0 cc.

After twenty-four hours, stains revealed that acid-fast bac-teria had been ingested by the protozoa of all cultures. Thepresence of acid-fast bacteria did not depress the numbers ofprotozoa nor cause observable damage or stimulation to them.The next day all cultures were transferred, 1 cc. of culture being


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added to 10 cc. of sterile medium. After five days, examinationrevealed that some, but only a very few, acid-fast bacteria werecontained in the protozoa. Fresh transfers were made as before.After five days examination was made again. No acid-fast bac-teria could be found. This indicated that multiplication of acid-fast bacteria did not occur within ciliates, even when the mediumin which the ciliates grew was very favorable for acid-fast strains.Jordan (1932) detected no evidence that tubercle bacilli multi-plied within Colpidium campylum.To determine the effect of ciliates on the acid-fast bacterial

numbers, pure cultures of the following composition were pre-paxed:

1. Gram-positive bacillus and Avian 5812. Gram-positive bacillus C. Steinii and Avian 5813. Gram-positive bacillus C. Steinii and M. ranae4. Gram-positive bacillus C. cuculus and Avian 5815. Gram-positive bacillus C. cuculua and M. ranae6. Gram-positive bacillus and C. cuculus

Medium D was used because it favored acid-fast bacteria andbecause the protozoa developed profusely in it when the tinyGram-positive bacillus was included. A sufficient population ofprotozoa could not be obtained when the tiny Gram-positivebacillus was omitted. Of the medium, 5 cc. amounts were inocu-lated with 1 cc. of desired culture without the acid-fast bacteriaand allowed to incubate for three days at 230C. To 5 cc. amountsof sterile medium1 1 cc. transfers were again made and allowedagain to incubate for three days so that the protozoa would benumerous. One cubic centimeter of a D medium, acid-fastsuspension was added to each culture as desired; the mycobac-teria had been dispersed by shaking with glass beads.Twenty-four hours later all bacteria were counted by a special

technic. An aliquot was taken and the Gram-positive bacillicounted by ordinary plating. Then to each suspension was added5 cc. of 2 per cent sodium hydroxide. Five minutes action waspermitted and the material was plated on crystal-volet-glycerol-agar. On these plates only acid-fast bacteria developed. M.ranae failed to form colonies so no counts were obtained for it.

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Each culture was duplicated. The counts are:Group Protozoa Gram-positive bacili Awian 631

1. (No protozoa)......0.........0/cc. 520,000,000/cc. 84,000/cc.2. (With C. Steinii). 2,0002 0,000,000 40,4004. (With C. cuculu) 1,000.39.01,000 39,000,00 9,200

The above technic was repeated to check results. Counts weremade 12 hours and 60 hours after the mycobacteria were added.The Avian 531 counts are given below.Time after Asian 581 Culture with Culture without

was added C. Steinii C. Steinii12 hours.................................. 162,000 660,00060 hours.................................. 1 1,600,000

An additional experiment conducted after experimental meth-ods of counting Avian 531 were more satisfactorily worked outrevealed that Avian 531 numbers changed from 15,000,000 to25,000,000 per cubic centimeter in C. Steinii and in C. cuculuscultures in two weeks. The plate count was the same for theculture without protozoa. The same Gram-positive bacillus usedin the previous experiments was included. Each count was basedon the average of 6 plates, and each count was duplicated. Itwas thus demonstrated with certainty that the protozoa did nothave a pronounced destructive action on this tubercle bacillus.The slight observed increase in Avian 531 is not of significantmagnitude to indicate an actual increase in Avian 531 numbers.There is generally an increase in the plate count of Avian 531following dispersion in a liquid medium, conditions being similarto those of this experiment.There was considerable evidence that an excessive concentra-

tion of tubercle bacilli in a medium was unfavorable to protozoa.When a very concentrated Avian 531 suspension was added toa culture of protozoa in a bacterial medium well suited for rapiddevelopment of the protozoa, the protozoa were decidedly in-hibited as evidenced by population. Thus, Avian 531 added inexcessive quantities to vigorous C. Steinii and C. cuculus cultureson A. aerogenes or other bacteria diminished the multiplicationrate of these protozoa. This is easily explained, for Avian 531is a poor food for C. Steinii as compared to many other bacteria.

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When the protozoa are induced to ingest vast quantities of thepoor food, the growth rate is diminished. Also, even a high con-centration of bacteria better suited as protozoa food may checkgrowth.

MORPHOLOGIC STUDIES OF THE ACID-FAST BACTERIA DURING

INGESTION BY CILIATES

With due consideration for the prevalent knowledge of cellulardigestion, it was deemed likely that marked changes in the mor-phology of the acid-fast bacteria would occur as a result of in-gestion of ciliates. The ingested bacilli could be readily studiedwhen stained by the acid-fast procedure. An extended study ofthe bacilli was therefore made.The bacilli occurring within C. Steinji grown upon pure cul-

tures of Timothy 1589 and Avian 531 were first observed. Largemasses of acid-fast bacteria were found in which the individualorganisms were often indistinguishable. This did not necessarilyindicate a homogenization of the bacilli to a mass of acid-fastmaterial, however, because whenever fewer bacilli occurred in aciliate, the bacilli appeared to be unchanged from those of theoriginal culture. The masses probably tended to appear homoge-nous because of the compact packing of the individual bacilli andbecause the decolorizer failed to remove the dye completely fromthe central portion of the ciliates. The tendency of dye reten-tion was frequently noted.Many observations of acid-fast bacteria were made at various

time-intervals following the addition of mycobacteria to an activepure mixed culture of a ciliate and the tiny soil bacillus. Theamount of mycobacteria suspension was so regulated that largemasses did not accumulate, the individual bacilli thus being dis-tinguishable within the protozoa. Some ingestion occurred im-mediately after addition of the acid-fast bacteria. During a weekof observations of Avian 531 cultured with C. cuculus and C.Steinit no morphologic changes were noted as a result of ingestion.Observations were rendered somewhat difficult by the pleo-morphismn of Avian 531. The bacilli varied in size and shapeand in degree of granulation. No morphological change has beenattributed to ingestion of mycobacteria by C. cuculus or C. Steinv.

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Isolation of pure cultures of a soil amoebaSoil amoebae were isolated for the purpose of studying the rela-

tion of this group of organisms to the acid-fast bacteria. Theisolation procedure is essentially the same as described bySevertzoff (1921).A particle of soil was placed on the center of a cross produced

on an agar plate by streaking a pure culture of Aerobacter aero-genes. The agar medium consisted of 100 cc. of nutrient broth,900 cc. of water and 15 grams of agar. Less agar was used bySevertzoff, but the amount of exuded water caused the undesir-able bacteria to spread too rapidly to the ends of the crosses.The plates were kept under bell jars at about 250C. An amoebawas soon noted on the plates. After about a week transfers weremade from the outermost portions of the crosses to the center ofother similar plates. Transfers were repeatedly made in thismanner. Microscopic examination of the plates revealed theprogress of the amoeba along the streaks. Transfers were al-ways made as soon as they reached the tip of the cross. A singleamoeba was picked on one occasion so that the strain would bepure. After seven or eight transfers the amoeba was found tobe in a pure culture of A. aerogenes as far as bacteriological studiescould be used to determine the purity. Stained smears and colo-nies were typical of the pure culture. None of the bacteria sur-vived toluol treatment for more than fifteen minutes.

It was necessary to free amoebae from the A. aerogenes inorder that the amoebae could be introduced into pure cultures ofacid-fast bacteria. Toluol treatment of the mixed culture wastried. The amoeba survived only for six minutes so this methodcould not be considered successful. Although other means couldhave been tried it was deemed best to seek a more resistant typeof soil amoebae. Normal soil was treated with toluol for threehours and used for the purpose of isolation. It was believed thatonly protozoa which form very resistant cysts would survive.Many amoebae survived and two distinct types were induced tolive upon pure cultures of A. aerogenes. Thirty minutes treat-ment with toluol destroyed the A. aerogenes and left the amoebacysts viable. These bysts could then be transferred, free from

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all contaminants, to any bacteriological medium or culturedesired.

Amoebae and mycobacteriaAmoebae and acid-fast bacteria were introduced on the same

agar plates to study the effect of the acid-fast bacteria upon theprotozoa. All the culturing was done on an agar medium con-taining 100 cc. of nutrient broth and 15 grams of agar per liter.The amoebae were carried with pure cultures of A. aerogenes.The amoebae were added to all plates in aqueous suspensionstaken from slant cultures. Acid-fast bacteria were added bymeans of a finely dispersed, filtered, aqueous suspension. Twodistinctly different types of soil amoebae and three mycobacteria,Avian 531, M. ranae, and Timothy 1589 were used.At first the acid-fast bacteria were added to the plates at the

time the amoeba A. aerogenes suspensions were added. It wasnoted that amoebae were much retarded by the mycobacteria.No desirable active cultures were procured. Amoebae and acid-fast bacteria were successfully grown together when the amoebaewere permitted to develop with A. aerogenes on an agar plate andthe mycobacteria added after this development had occurred.

Preparations for staining were then made. The amoebae weresuspended in 5 per cent acetic acid containing 0.1 per cent eggalbumin by allowing the solution to wash about the surface ofthe plate. This material was placed on a glass slide and per-mitted to dry slowly. The Ziehl-Nielsen technic of staining wasused. The conclusions drawn from the observations are:

1. Mycobacteria suspensions added to plates of agar mediumhindered the development of amoebae.

2. Acid-fast bacteria placed on agar plates with amoeba sooncollect about or within the amoeba cells.

3. Encystment is hastened by the mycobacteria.4. No fat globules were noted in the amoebae with or without

acid-fast bacteria, Sudan III being used as the fat stain; there-fore, no abnormal fat distribution was observed.

5. The mycobacteria may have hindered the amoebae in sev-eral ways. (a) By toxic products. (b) By acting as indigestible

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bodies which prevent normal feeding. (c) By a combination offactors.No very good evidence can be here presented for any of these

views. The acid-fast bacteria were not observably disintegratedby the amoebae. Therefore toxic products are somewhat diffi-cult to account for. The great number of mycobacteria groupedabout some amoebae indicated that these amoebae might havecollected sufflcent indigestible material to hamper normal digestionprocesses.

SUMMARY

1. Several methods were devised for conveniently and rapidlyintroducing pure cultures of protozoa, both ciliates and amoebae,into any pure culture of bacteria desired.

2. A culture of Colpoda Steinii was introduced into a nutrientbroth suspension of Avian 531. The ciliate multiplied and wascultured thus for more than one year. Nutrient broth alone orthe filtrate of a glycerol broth culture of Avian 531 did not sup-port growth of the ciliate.

3. Colpoda Steinii and Colpoda cuculus do not destroy Avian531, as evidenced by plate count studies.

4. Some acid-fast bacteria support growth of C. Steinii, othersdo not, a high degree of specificity being manifested.

5. Stains revealed large numbers of acid-fast bacteria to beingested by C. Steinii and C. cuculus.

6. Several strains of soil amoebae failed to grow upon pure cul-tures of acid-fast bacteria.

7. Excessive concentrations of acid-fast bacteria inhibit pro-tozoa.

REFERENCES

JORDAN, L. E. 1932 Jour. Infect. Dis., 51, 482-488.PETERS, R. A. 1921 Jour. Physiol., 55, 1-32.SEVERTZOFF, L. S. 1921 Centr. Bact., 92, 151-158.

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the relationship of soil protozoa to tuber- nature and

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