rhizobium'rhizobium species is limited, an abundance of discussion exists concerning...

THE STABILITY OF CULTURES OF RHIZOBIUM'LOIS ALMON AND I. L. BALDWIN

University of Wisconsin, Madison, IVisconsin

Received for publication October 26, 1932

The data to be presented here bear primarily on the relation-ships of six types of cultures, derived from clover or pea nodules,or stock cultures of Rhizobium trifolii or R. leguminosarum.These six types, while they were derived from sources whichmight lead one to suspect their identity with the clover andpea nodule organisms, nevertheless possessed cultural charac-teristics markedly different from those of the rhizobia. A num-ber were chromogenic, and some produced only negligible amountsof gum. These facts were considered worthy of further investi-gation, for they pointed to the possibility of radical changes inthe root-nodule organisms which, if properly understood, mighthelp to explain the sudden losses in infective power which areknown to occur and the fluctuations in nitrogen-fixing abilitywhen in association with leguminous plants.The transformations, one to another, of the six abnormal types

of organisms and of the normal type as treated in this paper,coupled with the presence of filterable forms, are suggestive ofdissociation phenomena as described by those who have workedwith other organisms. Previous citations of cultural variationhowever among the species of Rhizobium have been few. Withminor modifications, only one type of colony for each cross-inoculation group has been recognized. Deviations from thesetypes have usually been looked upon by other workers as con-taminations. The only unquestioned description of a rough formis that of Israilsky and Starygin (1930). No intermediates arementioned. Our cultures do not conform to their description ofa rough race.

' Published with the approval of the Director of the Wisconsin AgriculturalExperiment Station.

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20LOIS ALMON AND I. L. BALDW'IN

In view of the chromogenic character of certain of the culturesdealt with in this paper it is interesting to note that the contentsof lupine nodules in the hiands of Frank (1890) yielded yellowcolonies more thian once, and that the name Rhlizobiuwn leguiiwh0-sarum as applied by Frank was thus originally intended to cover

a pigmented form. A variety of chromogenic and other aberrantforms from lupine, Viciaafaba, and Trifoliurn incarnatnlum noduleswere also described by Gonnermann (1894). It is possible, in theliglht of the data to be presented in this paper, that soine of hiisorganisms bore some relationship to the true nodule formers,tlhough his reported ability to induce nodule formation withi thesecultures is questioned. Some of the types isolated by de R{ossi(1907) called by him contaminants, might also, conceivably, besimilar to some later to be described in this paper, since in certaiininstances these were the predominating types of organisms to beisolated from nodules. A red-browni form, according to i\Iilovidov(1928) is the usual inhabitant of the nodules of Carmichaelia.It is difficult to determine from the meager description givenwhether the Bacillus luteo-albus which was found by Beijerinck,(1888) in some nodules was like any of our cultures or not. Andit is not likely that any of the five species isolated in a number ofinstances by Burrage (1901) was like ours. Aside from thesereferences to possible aberrant types of the nodule organisms ofleguminous plants, the literature, as far as has been ascertained,is devoid of mention of anything that can be considered compara-ble to what is about to be described here. Only the reports ofIsrailsky and Starygin (1930) and of Milovidov (1928) giveadequate evidence for the identity of their cultures.And yet it should not be too surprising to find chromogenesis as

a characteristic of dissociating Rhizobium species. MIembersof the colon-dysentery-typhoid group, whiclh may be not fardistantly related to these organisms, have several times beencredited, on seemingly good authority, with this property; andit should be noted that the pigment reported is yellow, which isthe predominating one found in our cultures. Thus, Levy (1904)isolated a yellow, gas-producing organism, having most of thecharacteristics of Escherichia coli, from dough. H6vell (1916)

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STABILITY OF CULTURES OF RHIZOBIUM

obtained organisms producing yellow pigment, but reactingotherwise like Eberthella typhi from stools and urine; these organ-isms were not pathogenic. Kohlisch, 1916, likewise found yelloworganisms in feces and urine. By agglutination procedures hefound them to be related to paratyphosus B organisms. Alm-quist (1911) obtained yellow cultures from filtrates of E. typhiAnd Kendall (1932) obtained yellow cultures from filtrates of E.coli and from a culture of E. coli grown in his K medium.Though information concerning cultural variation among the

Rhizobium species is limited, an abundance of discussion existsconcerning morphological variation. An adequate review ofthis discussion has been prepared by Fred, Baldwin, and 1I\cCoy(1932). Suffice it to say here that much of it centers around thepossibility of a complex life cycle involving protoplasmic fusions,which, if adequately substantiated, would provide an explanationof the mechanism by which new forms might arise.

DESCRIPTION OF ORGANISMS

The six types of aberrant cultures treated are described belowfor purposes of citation. The type number, followed by a serialnumber, is used in the designation of individual cultures in thetables and in the text. (See table 1.) In cases in which noreference is made to particular cultures the key descriptions areused in the text. Both type numbers and key descriptions areused in the tables.The types are differentiated chiefly on their macroscopic

colony appearance on yeast-water mannitol agar (Fred and Waks-man, 1928). Occasionally a culture has seemed to belong in aposition intermediate between one of these types and the normaltype. Such cultures are referred to in the tables simply as"intermediates."

More complete descriptions of organismsType number andkey description

1 Thin, scanty, non-gummy growth on yeast-water mannitol agar.Colorless, Non-chromogenic. Small colonies. Behavior in gelatin andnon-gummy litmus milk variable. Long rods.

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TABLE 1Origins of the aberrant cultures

TYPE AND SERIALNUMBERS SOURCE

Colorless, non-gummy

1-1 Second plate, Hauduroy (1929) method, of lytic filtrate of R.trifolii, 235

1-2 Same source as 1-11-3 s.c. Transfer from R. trifolii, 205-1 s.c.1-4 s.c. Transfer from R. trifolii, 205B s.c.

Yellow, non-gummy

2-1 s.c. Pea inoculated with R. legurninosarum, 3132-2 s.c. First plate, Hauduroy method, of filtrate of R. trifolii, 205-1-2

s.c.2-3 s.c. Second plate, Hauduroy method, of filtrate of R. trifolli, 205-1-3

s.c.2-4 s.c. From R. trifolii, 2142-5 First plate, Hauduroy method, of lytic filtrate of R. trifolii, 2362-6 From R. trifolii, 205-1-3 s.c., grown in filtrate of same culture2-7 Same source as 2-62-8 Nodule of red clover

Wihite

3-1 s.c. Same source as 2-63-2 Same source as 2-63-3 From R. trifolii, 209

Yellow, gummy

4-1 s.c. Red clover inoculated with R. trifolii, 2344-2 s.c. Same source as 4-14-3 s.c. Same source as 4-14-4 Same source as 4-1

Brown, gummy

5-1 s.c. Second plate, Hauduroy method, of lytic filtrate of R. trifolii,236

5-3 s.c. Same source as 5-15-4 s.c. Same source as 5-15-5 s.c. Same source as 5-15-6 s.c. Second plate, Hauduroy method, of lytic filtrate of R. trifolii,

2355-7 s.c. Same source as 5-6

Pink

6-1 s.c. Plate colony, dissociant of 5-5

Cultures indicated by the letters "s.c." follow-ing the number were developedfrom single cell isolations.

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Type number andkey description

2 Scanty gum production. Lemon-yellow or orange pigment onYellow, yeast-water mannitol agar. Small colonies. Morphology

non-gummy variable- coccoid or rod-shaped, of the same order as the nor-mal organisms, but with the coccoid for predominating. Motile;single, polar flagellum. Behavior in gelatin and litmus milkvariable.

3 Like type 2, but white instead of yellow or orange. Growth inWhite gelatin, but no liquefaction after four weeks. No visible change

in litmus milk.4 Gummy to almost as great an extent as normal Rhizobium trifolii

Yellow, or R. leguminosarum. Yellow chromogenesis. Morphologygummy like the normal organisms. Growth on gelatin; no liquefaction

after four weeks. Some cultures produce a serum zone in litmusmilk.

5 Like type 4, except for the color of the pigment produced. ThisBrown, varies with the age of the culture from a brownish yellow to agummy dark yellowish, bluish, or reddish brown, or, at times, purple.

6 Scarcely gummy at all. Chromogenesis, a bluish-pink. Pin-Pink point colonies on yeast-water mannitol agar. Thrives some-

what better on plain agar. Rods-somewhat broader and moreuniform in size than the typical organisms. Grows in gelatin,but does not liquefy it in four weeks. Produces no visiblechange in litmus milk.

These cultures do not form nodules on clover or alfalfa plantsunder greenhouse conditions which are favorable for noduleproduction by typical cultures. On conventional laboratorymedia they are as stable as the normal Rhizobium cultures.

Evidence bearing on the identity of the six types of organismsjust described has been gathered along three general lines: thederivation of atypical forms from typical cultures; the partial ortotal changing of one aberrant type to one or more others, withreversion to the normal form in two instances; and the relation-ships betrayed by specific agglutination.The normal cultures mentioned in the text have all been

carried for more than a year. They have shown no evidence ofimpurity by the milk and potato tests; and have shown themselvescapable of producing nodules when inoculated onto the properplants.A list and brief history of the aberrant cultures used is given

in table 1. For the reader's ready information, cultures of singlecell origin are designated in most of the tables by the letters "s.c."following the number.

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LOIS ALMON AND I. L. BALDWIN

THE DERIVATION OF ABERRANT CULTURES FROM NORMAL ONES

Accidental occurrence in nodules and stock cultures

It has previously been mentioned that the abnormal types withwhich we are concerned have been derived both from nodules andfrom stock cultures. In most instances they have been en-countered in cultures which underwent special treatment. How-ever, in a few instances they have been obtained from typicalcultures and from nodules in the normal course of their handling.

Usually, only occasional colonies from the plating of nodulecontents have shown aberrant characteristics, and these, as faras has been noted, have been of the colorless, white, or yellowtypes. (See table 1, culture 2-8.) In the plating from whichour yellow gummy cultures were derived, however, no other typeappeared, not even the normal type. And the nodules from whichthese platings were made had been secured in such a way thatpossibility for contamination was reduced to a minimum. Redclover seeds subjected to treatment with mercuric chloride andcalcium hypochlorite to free them from micro6rganisms wereplanted in Crone's agar (Bryan, 1922) in bottles stoppered withcotton. The only microorganisms in the bottles, supposedly,were those of the culture of phage-sensitive Rhizobium trifoliiintroduced for inoculation purposes. The platings from thecontents of the nodules formed under these circumstances in oneof the bottles were made after two and one-half months, andrevealed, as has been mentioned, only yellow, gummy colonies.(See table 1, cultures 4-1 to 4-4 inclusive.)2The cultures which first suggested the possibility of their

relationship with true Rhizobium were some of the yellow, non-gummy type which came from the multiplication of single cellsisolated from pea nodules. (See table 1, culture 2-1.) Thesenodules, it is true, could have contained organisms other than thenodule formers, since the plants from which they came were grownin open pots. On the other hand, the sand had been autoclavedlong enough to kill all organisms, and the usual precautions for

2 Type 4 cultures have also been isolated from nodule suspensions by single-cell technique.

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maintaining sterile conditions, such as the mercuric chloride andhypochlorite treatment of the seed, the use of sterile water,and the maintenance of humid conditions in the green-house, wereexercised.The occurrence of aberrant types among laboratory cultures is

sporadic, as it is among cultures direct from nodules. Upon afew occasions the bottles containing the organisms for inoculatingpurposes as they are sent to farmers have shown homogeneousgrowth of either of the yellow types. Twice, in the author'sexperience, one of duplicate transfers made from slants of single-cell clover cultures onto new slants of the same yeast-watermannitol agar has shown uniform growth of the non-gummyyellow type, probably incapable of forming nodules, while theother of the duplicate transfers has retained its original charac-teristics. The colorless non-gummy type has also been knownto appear in this way from single-cell cultures. (See cultures 1-3and 1-4, table 1.)

FilterabilityAlthough under seemingly uniform conditions abnormal cul-

tures are derived from normal cultures only fitfully and un-reasonably, under some controlled conditions they may appearmore regularly. In a high percentage of cases the filterable ele-ments in liquid cultures will give rise to cultures such as have beendescribed above. This has been determined by the cultivationof filtrates on agar plates by what is essentially the Hauduroy(1929) technique, which consists in spreading 0.5 cc. of filtrateover the surface of hardened agar in a Petri dish, incubating fora suitable length of time, washing off the surface with sterileliquid medium if the plate shows no growth, and transferring thewashings to a sterile agar plate, the washing and transferring tobe repeated seven times or more if growth does not appear earlier.For these organisms five days were allowed to elapse betweeneach washing. The medium used here, as in all other work withthese organisms unless otherwise specified, is the yeast-watermannitol medium in Fred and Waksman (1928). Berkefeld Nfilters were used. They were tested for their filtering efficiency

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with broth cultures of Serratia marcescens following eaclh twofiltrations of the experimental cultures.Table 2 gives the results of some of the attempts at cultivation,

witlh the essential data concerning the souir(ce of the filtrates.The cultures before filtration were grown in a modification of the

TABLE 2Results of cultivation of filtrates of normnal cultures of Rhtizobiui

NUMBERCULTURE OF AGE OF FILTRATE LYTIC TYPES OF GROWTH BY HAUDUROYNUMBER FILTRATES ACTIVITY METHOD

TESTED

days200-1 s. c. 2 42 - Brown, gummy205-1-2 s.. 2 35 - Yellow, non-gummy205-1-3 s.C. 2 42 - Yellow non-gurnlfy and

wNhite205B s.c. 1 28 - Colorless, non-gummy214-3 s.c. 3 4, 7, 28 - None218* 1 12 + Normal234 1 49 - t Colorless, non-gummy235 9 14+, 28, 33, 56 + Elch of the a)normal types

except pink236 5 28, 35, 56 + Each of the abnormal types

except pink236 1 28 -t White332 5 28, 84, 122, 150 + Colorless non-gummy,

white, yellow gummy, andnormal

* The filtrates of this and all of the cultures following it in the table wereobtained followiing lysis hy a stock bacteriophage.

t The filtrate control contained a granular growth like that describ)ed byHauduroy, 1929, as occurring in filtrates. This may account for the loss of lIvticactivity.

+ This sample yielded no growth by the Hauduroy method.

usual medium; the mannitol was omitted and the yeast-watercontent increased to 200 cc. per liter. Some were dissolved byphage activity before filtering. In the successful cases culturesappeared on the Hauduroy plates before the seventh washing,many of them as early as the first plate, although the correspond-ing tubes of filtrate showed no signs of growth, except in two cases,either within the original tube or when transferred to new tubes of

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liquid medium. Nor did microscopic examination of the clearfiltrates reveal the presence of any recognizable bacterial elements.Some of the filtrates were concentrated by evaporation, underpartial vacuum at relatively low temperatures, to facilitate suchexamination. The granular growth reported in two instancesconformed very well to Hauduroy's (1929) description of growthin filtrates, and to Hadley, Delves, and Klimek's (1931) descrip-tion of the "G" form.Of the thirty-two filtrates which were subjected to this method

of cultivation only four yielded no cultures at all and three ofthese had stood in the laboratory for a shorter period since filtra-tion than had those which yielded positive results. The ageof the filtrate may therefore be important in securing positiveresults by this method. Three of the filtrates gave rise to normalcultures; the remaining twenty-five showed abnormal forms on theHauduroy plates.

It is of interest that more than one abnormal type of culturedeveloped in some cases from a single sample of filtrate, but thatin no case did an abnormal culture and a normal one appear onthe same plate. We shall not try to explain these occurrences.Suffice it to say that the results seem to indicate that from normalcultures of Rhizobium trifolii and R. leguminosarum, some of themof single-cell origin, filterable elements may be developed withor without the action of a bacteriophage, and that these may giverise to normal cultures or to one or more of five abnormal types.Cultures 1-1, 1-2, 2-2, 2-3, 2-5, and all of the brown, gummytype listed in table 1 were derived by this method. The normalcultures obtained by this method have been tested for theirnodule producing capacity and found positive.

Filtrates of old cultures as mediaThe literature shows that dissociation often occurs among

bacteria when the environment is modified in such manner as tomake it unfavorable for the continued growth of the original form.It was thought that if cultures were allowed to grow in a liquidmedium for a period of a month or more, the medium would beso depleted of nutriment and so vitiated by the accumulation of

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238 LOIS ALMON AND I. L. BALDWIN

toxic products as to render it very unfavorable for the initiationof growth. Accordingly, the organisms were removed by filtra-tion from such old cultures and fresh inoculations were made intothe filtrates. The type of growth following inoculations intomedia thus modified appear in table 3. The cultures developingare represented in table 1 by cultures 2-6, 2-7, 3-1, and 3-2. It

TABLE 3Type of growth obtained followintg inoculation of normnal, single cell cultures into

filtrates of old single cell cultures

CULTURE CULTUREINOCULATED FROM WHICH

INTO FILTRATE MEDIUM TYPE OF GROWTH OBTAINEDFILTRATE DERIVED

209-2 5-3 Bean-extract sucrose Yellow, non-gummy209-2 209-2 Yeast-XNater* Yellow, non-gummy214-3 214-3 Yeast-water Yellow non-gummy and

white214-3 214-3 Yeast-water mannitol Normal205-1-3 205-1-3 Yeast-wN-ater Yellow non-gummy and

white205-1-3 205-1-3 Yeast-water Yellow, non-gummy214-1 214-1 Yeast-water mannitol Colorless, non-gummy214-1 214-1 Yea.st-water mannitol Colorless, non-gummy200-1 201-1 Yeast-water mannitol Normal200-1 200-1 Yeast-w-ater mannitol Yellow non-gummy and

white200-1 200-1 Yeast-w^ater Colorless, non-gummy107A 107A Yeast-water mannitol Normal107A 107A Yeast-water mannitol Normal107A 107A Yeast-water Normal313A 313A Yeast-water mannitol Yellow, gummy313A 313A Yeast-water mannitol Normal

* Yeast-water, 200 cc. per liter in addition to the usual inorganic salts.

cann.ot well be determined, of course, whether the cultures arisingin the filtrates after reinoculation are the result of the multiplica-tion of the new inocula or the further development of the filterableelements from the previous cultures. In order to obviate thelatter situation as far as possible the cultures grown for filtrationto yield the vitiated medium were, in a few cases, at least, chosenfrom those which lhad previously given rise to no growth fromtheir filtrates, a fact which might lead one to surmise that filter-


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able living units were present only sparingly, if at all. (Cultures214-1, 214-3 and 313A.) As further control over the possibilityof the filtrates themselves giving rise to new cultures, one-half ofeach filtrate was always retained without reinoculation and aportion of it plated at the same time that the inoculated portionwas plated. It should be emphasized that filtrates do not ordi-narily yield cultures in plates by any but the Hauduroy technique.This control work was done by the ordinary plating procedure inwhich the inoculum is incorporated into the melted agar.One culture each of the crown-gall and hairy-root organisms,

used for comparison in this work, showed no change when grownin their homologous filtrates. The four trials with these organ-isms are, of course, insufficient to justify definite conclusions.

Action of bacteriophageNo organized effort has been made to discover the extent to

which an active race of bacteriophage may encourage the appear-ance of aberrant types. In the hundreds of cultures whichhave been subjected to the action of the phage, however, bothyellow types have appeared frequently, usually as secondarygrowths, though sometimes before lysis was manifest. Thesetypes could be detected in the liquid medium by the pronouncedmilky turbidity which they produced. Tubes showing this typeof turbidity were usually set aside for confirmatory transfer ontoslants of the usual medium. In one series of eighteen tubes fiveshowed the presence of the non-gummy, yellow type, but usuallythe ratio of aberrant to typical cultures was much lower than that.The yellow types have also appeared on agar plates which hadbeen prepared to show plaque formation. On some of theseplates plaques were present, but the growth was a marbledmixture of the typical and the yellow. It might be added that theyellow types have shown no indication of being sensitive to thetwo races of phage used here.

Growth in soilIn order to render soil fit for pure culture work, it must, of

course, be freed from the life existing in it. The long heating

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necessary to do this may alter it so as to make it quite differentfrom an ordinary soil habitat. But whether or not results ofgrowth of Rhizobium in autoclaved soil may be interpreted asbeing similar to what would occur in nature, they offer additionalevidence for the appearance of hitlherto unrecognized types fromcultures of single-cell origin.The soil used was a garden loam containing a considerable

quantity of humus. To one-half of it calcium carbonate waIsadded to make the reaction more favorable for the organisms.Both lots were sterilized in test tubes for twelve lhours at 15pounds pressure, whereupon transfers were made to brothi to

TABLE 4

Forms derivedfronm normiial cultures in soil by plating it yeast-water mnannitol agar

SOIL WITHOUT CaCO3 SOIL WITH CaCO3CULTURE _

First month Second month First monthl Second month

200-1 s.c. All normal 1 colony, yel- All normal All normallow gummy

209-2 s.e. Some l)rown, A few brown, All normal All normalgummy gummy

214-4 s.c. All yellow, All yellow, All normal All normalgummy gummy

107A s.c. All normall All normal All normal All normal313B s.c. All normal All white Some colorless, All normal

non-gummy

check the sterility. No growth was observed during a two-weekperiod. After inoculation with liquid cultures the soil was keptmoist. Plate cultures were made at the end of one and twomonths. The results appear in table 4.The most striking thing shown by this table is that the aberrant

forms appeared most often in the acid soil. Indeed, scarcely anychange was noted in the cultures which were kept in the soil towhich calcium carbonate had been added. The amount of clhangewhich occured in the acid soil is not so great as might be supposedat first glance. Only two cultures lost their normal characteris-tics completely. One did not change at all, and another showedonly one aberrant colony on plating; while the remainiing cultureshowed a small percentage of the brown, gummy type.

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Growth in other mediaThe use of yeast-water medium (200 cc. per liter; no mannitol)

rendered alkaline to a pH of 8.2 resulted in the appearance ofsome of the atypical forms, chiefly the white and non-gummyyellow. The same medium at a pH of 7.6 encouraged the appear-ance of type 4 from three normal single-cell cultures. Thenumber of cultures which gave evidence of change in these media,however, was a very small percentage of the number which havebeen grown in them.

Nodule contents of plants kept in darkThe plants, mentioned previously, which contained only the

yellow, gummy type in their nodules were more than two monthsold, and quite yellow. It was postulated that carbohydratestarvation might have been responsible for the abnormal appear-ance of the nodule cultures. To test this hypothesis two bottlescontaining nodulated red clover plants eight weeks old were keptin a dark 30°C. incubator for eight days. At the end of this timethe nodule contents were plated, with parallel plates from nodulesof plants of the same age which had received normal daylightduring the same period. Only typical colonies were obtainedfrom the two lots of nodules.

CHANGES AMONG ABERRANT CULTURES

If the aberrant forms, which may be caused to appear so readilyfrom cultures which show only typical characteristics, could becaused to revert to the typical state there would be little doubt ofthe relationship of these forms to the typical form. Even thedemonstration of relationship of the aberrant types to one anothermight be considered to support the hypothesis of the commonorigin of all of them. A number of means have therefore beenemployed to attempt to hasten reversion or change from one formto another. It should be emphasized that under the ordinaryconditions of transfer over a period of more than a year each typehas retained the characteristics which it had when first isolated,except in the cases listed in table 5 in the column headed "Stock."The methods employed, consisting primarily in modifications

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of media, were of the types which other workers lhave found effec-tive in causing dissociation. Particularly were adaptations ofmethods used which others had found successful in causing rough

TABLE 5New types arising from aberrant cultures after growth in v,arious media

CULTURE MEDIUIM

CULTURE Bean-extract Stock; yeast-NUMBER Reduced iron Dead cells Soil sucrose water mannitol

1-2* 0G form*1-2*1-4 s.c. 3 Normal2-1 S.C.3 4 and 52-2 S.c.1,2,4 Intm. t 3 3 and Intm. Intm. Intm.2-3 s.c. 5 3 62-4_s.. 3 5 3 and 62-5' 12-7 1 and 62-8' 3 23-2 24-1 s.s. 2 5 54-2 s.c. 5 5 25-5 s.c.6 65-6 s.c. 3 Intm.5-7 s.c. Intm.6-1 s.c. 3

* Hadley, Delves, and Klimek, 1931.t Intm. = intermediates, approaching the normal form in appearance.I Repeated transfer gave rise to type 3 from cultures 2-2 and 2-8 and to inter-

mediates from culture 5-1.2 Culture 2-2 produced some intermediates in peptone sucrose liquid. Types 3

and 6 appeared in culture 2-5 in this medium.3Type 4 appeared in culture 2-1 when treated by the Quirk method.4Type 3 appeared in culture 2-2 grown in beef-peptone broth.' Culture 1-1 showed only normal colonies after growing in a filtrate 209-26 Culture 4-4 gave rise to types 2 and 3 on potato agar. Type 6 appeared in

culture 5-5 when grown on this medium.For key to types see page 231.

cultures to revert to the smooth form, for while our aberranttypes may not be truly rough, neither are they truly smooth, ifthe typical form be considered the only truly smooth one.Among these methods, adaptations of the repeated transfer


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procedure used by Koser and Styron (1930) and the one involvingdilution of organisms and pH adjustment developed by MissQuirk (1931) yielded no truly positive results. A few changes intype occurred which are noted in footnotes 1 and 3 to table 5.Growth in peptone sucrose medium, beef peptone broth ad-

justed to pH 5, 7, and 8, and potato agar was no more successful.See footnotes 2, 3, and 6 to table 5.

FiltratesIn view of the success attendant upon the growth of normal

cultures in filtrates of old cultures with regard to their productionof abnormal types, the reverse was attempted. Filtrates ofnormal culture 209-2 were used entirely. Success in instigatinga reversion to the typical form was attained with culture 1-1 acolorless, non-gummy type, on two occasions. Seven othercultures, representing the remaining five aberrant types, showedno change.

Reduced ironThat the oxidation-reduction potential of the medium has an

important effect on the ability of aerobic organisms to initiategrowth is known from the studies of Allyn and Baldwin (1932).If it is indeed such a potent factor in the environment, it mightbe expected to induce changes in organisms if altered in such afashion as to make the environment markedly different from theusual. Rhizobium, at least the two species which are beingconsidered here, appears to have as an optimum a potential inthe vicinity of the value furnished by the yeast-water mannitolmedium, but will grow normally in media varying to some extenton either side of this optimum. The genus is not generally con-sidered to be anaerobic, nor does it ordinarily do well in veryreduced media. Therefore, anaerobic or very reduced conditionsmight be expected to supply sufficient shock to the organisms tocause them to alter their physiology and consequently theircultural characteristics if they survive at all.One method of supplying an environment relatively low in

available oxygen was the addition of iron reduced by hydrogen to

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yeast-water mannitol medium. Fourteen cultures were sub-mitted to this treatment, and plated after one week on the ordi-nary unreduced medium. Of these, only five (see table 5) gaveany readily detectable evidence of having been influenced by theunsuited environment, but the evidence was marked enough tobe significant. From 20 to 40 per cent of these five cultures wereof the new types.

Dead cellsThe most outstanding examples of transformation of rougl

into smooth types have been accomplished with the pneumococci,often with a change of specific type involved. Transformationsof this nature have been brought about both in vivo (Griffith,1928) and ini vitro (Dawson and Sia, 1931), by growing them incontact with dead smooth cultures. Dead cultures of 205-1were employed for our attempts with these organisms as suggestedby the in v,itro experimenlts of Dawson and Sia (1931). Thenormal culture was grown in liquid medium for four days. Thetubes were then immersed in a 70°C. water bath for fifteen minutesto kill the organisms. Following this they were centrifuged andthe supernatant liquid was removed. Transfers from the sedi-ment onto slants were made at this time as a control on the effec-tiveness of the killing procedure. Then fresh medium was added,and inoculations of atypical cultures were made. Since dilutionis important in the case of the pneumococci, it was thought ad-visable to dilute these organisms also. Accordingly, a needletransfer from young liquid cultures was made to 10-cc. tubes offresh medium, and a loopful of this was used to inoculate themedium containing the dead cells. Thirteen cultures were soinoculated in duplicate on two separate occasions. Platings weremade after four and eight days. The changes are reported intable 5.A higher percentage of cultures showed change after having

grown in this medium than in any other except soil. Five of thethirteen gave marked evidence of change of type, and one other(culture 5-6) gave indication of dissociation to the white type, inthat one colony of this type appeared on one of the plates.

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SoilIf the forms which we have succeeded in isolating in the labora-

tory occur in nature-and some of them do, in nodules, as wehave seen- they should logically be found in the soil as well.Further reasoning would lead one to expect any changes whichoccur naturally to go on most readily in a natural environment.With this in mind, aberrant cultures were kept in garden soil(autoclaved for twelve hours) for four months. The soil wasrather rich in humus and had an acid reaction. This was remediedby the addition of calcium carbonate to one lot, for acid soil is ofdoubtful suitability for the clover organism. The new typesarising by this method are listed in table 5.

Fourteen cultures were so carried. Eight of these showed thepresence of one or more additional types after four weeks or morein the soil. The tendency seemed to be toward the appearanceof the brown, gummy type in cultures of either of the yellowtypes. The brown, gummy type, itself, was stable in soil inthose cases in which it lived; but in the acid soil no evidence ofthe presence of the cultures of this type could be found afterthree months. In other cases the reaction of the soil seemed tohave little or no effect upon the viability of the culture or thetype of change produced.

Bean-extract sucrose mediumA bean-extract sucrose liquid (Fred and Waksman, 1928) was

quite successful from the standpoint of encouraging changes.Twelve cultures were kept in this medium for one month. Plat-ings were made after three and twenty-eight days. Five of thecultures (see table 5) showed evidences of change.

StockA statement was made earlier in this paper to the effect that

the abnormal cultures are as stable under laboratory conditionsas are the normal ones, if not more so. In spite of this fact rever-sion to the normal form did occur in one culture of the colorless,non-gummy type. Culture 1-2 showed gumminess in the stockwhich had been kept in the ice-box for a month. It was plated

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and found to consist of approximately 75 per cent typical colonies,five of which were fished to slants and used for plant inoculationwith resulting nodule production. Likewise, culture 5-6 (brown,gummy) showed characteristics of the normal type in stock. It,too, yielded a high percentage of normal-looking colonies onplating. These cultures were non-effective, however.The only case of change in a type 3 culture occurred in a stock

which had received no special treatment. Culture 3-2 (white)showed patches of non-gummy, yellow growth.One of the few cases in which the pink type arose from the

yellow, non-gummy type was also of this kind. Culture 2-1showed discrete colonies of the pink type scattered throughoutthe confluent growth of type 2 on the slant.

IntermediatesThe cultures reported as "intermediate" in table 5 appeared,

in most cases, to be mixtures of the normal form and one of theabnormal ones which could not be separated from each other byplating. But since such cultures caused no nodule formation onclover or alfalfa plants, probably no truly normal component waspresent. A few of the intermediates appeared perfectly normal,but because of their inability to form nodules have still beenlisted as intermediates. This is true of the ones derived fromculture 5-7 (brown, gummy) in soil and from culture 5-6 as it wascarried in stock.A few attempts were made to cause these various intermediates

to change further by special manipulation. However, repeatedtransfer at short intervals in liquid medium, growth in soil, growthin homologous immune serum (10 per cent), and growth in amedium containing sodium hyposulphite effected no completereversions to the normal type.The fact that each of the aberrant types was secured from some

of the other aberrant types and that each of the aberrant types-in several cases single-cell cultures-gave rise to one or more ofthe other aberrant types furnishes strong evidence to support thehypothesis that all of these aberrant forms are related. Theproduction of the normal infective form from culture 1-1, a

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colorless, non-gummy and non-infective type, and the productionof intermediates, cultures approaching the normal in form butnon-infective, from several of the aberrant types indicates therelationship of these aberrant types to the normal type ofRhizobium.

RELATIONSHIPS SHOWN BY SPECIFIC AGGLUTINATION

Agglutination tests yielded no conclusive results. Some agglu-tination of organisms of one type was caused by antisera ofanother type, but there was little regularity in the groupingtendencies thus shown. The strongest evidence of relationshipwas shown among cultures 1-3, 3-1, and 6-1. Cross-agglutinationreactions occurred quite consistently with these cultures, theonly flaw being that the antiserum for culture 3-1 caused aggluti-nation of culture 1-3 in a titer no higher than 20. This is not aserious flaw, however, for the titer of this antiserum against thehomologous organism was only 80. No normal agglutinins forthese organisms could be detected in the sera of any of therabbits used.

DISCUSSION

It may be seen from the foregoing paragraphs that filtrates oftypical Rhizobium tr2folii if spread upon agar plates and carriedfrom plate to plate in series may give rise to one or more of sixrecognizedly different types, only one of which is like the original.A single culture may give rise to as many as four different types.This has occurred in a sufficient number of cases and with ade-quate enough control, it is believed, to warrant assumption of theexistence of filterable elements in such typical cultures. Thesefilterable elements, in the majority of cases, develop into culturesnoticeably different from the ones which had been filtered. Andyet some of the typical cultures which were so filtered representthe multiplication of single cells isolated by the modified Cham-bers micro-manipulator.Only two explanations present themselves in an attempt to

elucidate these facts. Either the pure cultures of Rhizobiumtrifolii produce filterable bacterial forms capable of developing

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into cultures differeint from the parent, or, invisible bacterialelements of one or several contaminating species were picked upat the time of isolation of the single cells and multiplied unnoticedduring the subsequent history of the cultures up to the time of thecultivation of the filtrates.The evidence presented by the results of the other experimental

work is scarcely compatible with the second explanation. It isdifficult to see, for example, why invisible elements of six otherspecies of organisms should be so regularly associated with theroot-nodule bacteria. If these six others are to be interpreted asdifferent forms of one, it should be equally easy to conceive of thetypical form as being related and to call the seven types of culturesdifferent manifestations of the possibilities inherent in one species.That some, at least, of the six forms are related to one another

and also to the typical form would seem to be indicated by theagglutination tests.

Also, the ease with which cultures of one or another of theaberrant types may throw off one or more colonies of one of theothler types or become completely transformed into another type(as occurred with culture 2-3 in the medium containing deadnormal cells and with cultures 2-2 and 2-3 in soil) can conceivablybe interpreted as giving evidence of the interrelationships of theseforms. A condensation of the results given in the tables concern-ing changes in atypical forms shows that

The normal and white types have arisen from the colorless, non-gummy type.

The white, yellow gummy, brown gummy types and intermediateshave arisen from the yellow, non-gummy type.

The yellow, non-gummy type has arisen from the white type.The yellow non-gummy, white, and brown gummy types have arisen

from the yellow, gummy type.The white and pink types and intermediates have arisen from the

brown, gummy type.The white type has arisen from the pink type.

An interesting observation which may or may not be significantis that all of the types at one time or another have shown elements

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of the white type, but that this type itself is rather more stablethan any of the others.

In conclusion, it seems desirable to emphasize that although ourcultures under some conditions have been shown to be unstable-whether due to changes in the existing organisms or to newlyacquired dominance of elements hitherto masked does not matter-the factors which stimulate the manifestations of instabilityhave not been clearly delimited.

SUMMARY

1. Six culture types markedly different from Rhizobium trifoliiin its typical form have been isolated from cultures of this organ-ism under conditions which have reduced opportunity for outsidecontamination to the minimum.

2. Five of these types have been obtained by the Hauduroymethod of cultivating filtrates in series on agar plates. Bothlytic and non-lytic filtrates gave rise to cultures.

3. Four of the types were obtained by growing single cellcultures of the normal organism in a medium previously vitiatedby the long-time growth of the same or other cultures and freedfrom the primary growth by filtration.

4. All of the aberrant types have been shown capable of givingrise to one or more of the other types.

5. In two instances, cultures of a colorless, non-gummy typehave reverted to the normal form and regained their power ofinducing nodule formation on clover plants. A brown, gummytype has given rise to cultures which appear normal but do notform nodules.

6. Agglutination tests indicate some group relationship amongthe various aberrant types and the normal form.

REFERENCESALLYN, W. P., AND BALDWIN, I. L. 1932 Jour. Bacteriol., 23, 369-398.ALMQUIST, E. 1911 Centbl. Bakt. (etc.), I Abt., Orig., 60, 167-174.BALDWIN, I. L., AND FRED, E. B. 1929 Jour. Bacteriol., 17, 141-150.BEIJERINCK, M. W. 1888 Bot. Zeit., 46, 726-735; 767-771; 781-790; 797-804.BRYAN, 0. C. 1922 Soil Sci., 13, 271-287.BURRAGE, S. 1901 Indiana Acad. Sci., Proc. 1900-01, 157-161.DAWSON, M. H., AND SIA, R. H. P. 1931 Jour. Expt. Med., 54, 681-699.

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FRANK, B. 1890 Landw. Jahrb., 19, 523-640.FRED, E. B., BALDWIN, I. L., AND McCoY, E. F. 1932 Root nodule bacteria

and leguminous plants. Univ. of Wis. Studies in Science, No. 5, 343 pp.FRED, E. B., AND WAKSMAN, S. A. 1928 A Laboratory Manual of General Micro-

biology. McGraw Hill Book Co., New York, 145 pp.GONNERMANN, M. 1894 Landw. Jahrb., 23, 649-671.GRIFFITH, F. 1928 Jour. Hyg., 27, 113-159.HADLEY, P., DELVES, E., AND KLIMEK, J. 1931 Jour. Inf. Dis., 48, 1-159.HAUDUROY, P. 1929 Les ultravirus et les formes filtrantes des microbes. Mas-

son & Cie., Paris, 392 pp.HOVELL, H. 1916 Centbl. Bakt. (etc.), I Abt., Orig., 77, 449-452.ISRAILSKY, W. P., AND STARYGIN, L. 1930 Centbl. Bakt. (etc.), II Abt., 81, 1-11.KENDALL, A. I. 1932 Science, N.S., 75, 295-301.KO$HLISCH. 1916 Centbl. Bakt. (etc.), I Abt., Orig., 78, 136-141.KOSER, S. A., AND STYRON, N. C. 1930 Soc. Exp. Biol. and Med. Proc., 27, 978-

979.LEVY, F. 1904 Ztschr. Hyg. u. Infektionskrank., 49, 62-112.MILOVIDOV, P. F. 1928 Centbl. Bakt. (etc.), II Abt., 73, 58-69.QUIRK, A. J. 1931 Science, N.S., 74, 461.DE RossI, G. 1907 Centbl. Bakt. (etc.), II Abt., 18, 289-314.WRIGHT, W. H., AND NAKAJIMA, H. 1929 Jour. Bacteriol., 17, 10-11.


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rhizobium'rhizobium species is limited, an abundance of discussion exists concerning...

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