Degeneration and Regeneration of Streptomyces Griseus'ANNA MARIA WILLIAMS AND ELIZABETH MCCOY

Department of Bacteriology, University of Wisconsin, Madison 6, Wisconsin

Received for publication, July 13, 1953

An important consideration in handling industrialcultures is the effect of artificial media upon themaintenance of stability. Reese et al., (1949) investi-gated stability of penicillin production by Penicilliumchrysogenum Wis. Q176 when transferred in severalseries on slants of different agar media. In a total of403 transfers they observed only two sectors, onegiving a normal yield of penicillin and the other onlyhalf as much. They concluded that the strain Wis.Q176 can safely be carried by successive agar transfersat weekly intervals. No similar study on stability ofantibiotic production by a streptomycete has beenreported.The present study deals with (1) morphological

variation and stability of antibiotic production uponserial transfer of Streptomyces griseus on artificialmedia, (2) possible correlation of changes in potencywith changes in morphology, and (3) regeneration ofantibiotic-producing ability.

MATERIALS AND METHODSThree descendant strains from Dr. Waksman's

#9 strain of S. griaew (C, V, and an asporogenousvariant N) and four from his #4 strain (C-57, C-131,M366P1, and W4-2) were used. All cultures are com-mercial strains except W4-2, our* laboratory strain.The greater part of the work was done with M366P1.Ten replicate slants of yeast-glucose agar were inocu-lated from stock cultures of this strain, and wereconsidered as the original culture in the comparativeassays of antibiotic activity.The two long series of transfers were made on yeast-

glucose agar (1 per cent yeast extract, 0.5 per centglucose (cerelose), 0.1 per cent K2HPO4, 1.5 per centagar). This formula, or slight variations of it, is acommonly used medium for S. griseus. Shorter seriesof transfers were made on maltose and starch sporu-lation agars (1 per cent maltose or soluble corn starch,0.5 per cent tryptone, 0.05 per cent K2HPO4, 0.05 percent NaCl, 0.01 per cent FeSO4, 2.0 per cent agar).Cultures on yeast-glucose agar were transferred everytwo days and on maltose and starch agars every threedays, the interval being determined by the time neces-

1 Published with the approval of the Director of the Wis-consin Agricultural Experiment Station. This work was sup-ported in part by a grant of funds from the Upjohn Co., Kala-mazoo, Michigan.

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sary for good sporulation. A loopful of spores (or aloopful of surface growth with nonsporulating cultures)from the middle of the previous slant was spread up,down, and up the slant with a side-to-side motion.Incubation was for 5 days at 30 C, after which slantswere stored in an icebox at 6 to 12 C until tested.

Cultures were observed macroscopically at eachtransfer, and about every 30 transfers the slants werestreaked on plates of yeast-glucose agar by a standard-ized procedure and the resulting growth examinedmacroscopically and microscopically at various stagesof growth.To determine antibiotic production, duplicate or

triplicate 500-ml Erlenmeyer flasks containing 100ml of Waksman's fernentation medium (Waksman,1950) were inoculated from five-day-old cultures onyeast-glucose agar slants, the slants having beeninoculated from the serial transfers held for antibioticproduction studies. Flasks were incubated at 30 C ona rotary shaker, and a ten ml sample was removedaseptically on the 3rd, 4th, 5th, and 6th days for pHdetermination and microscopic examination of wholebroths and antibiotic assay of supernatants fromcentrifugation. The assay procedure was a dippingmodification of the standard paper disc-agar platemethod of Loo et al., (1945), with Bacillus subtilisas the test organism. One unit was equal to 1 micro-gram of pure streptomycin base.To obtain a spore inoculum from poorly-sporulating

cultures in one of the attempts at regeneration, afew loops of surface growth were suspended in about10 ml of sterile distilled water. The suspensions werecentrifuged under conditions of speed and time whichwould throw down vegetative growth but leave someof the spores floating. Spores were pipetted from thesurface and diluted in sterile 1:10,000 castile soap (aconcentration found to be non-toxic), and 0.2 ml wasspread on the surface of yeast-glucose agar plates.Dilutions were prepared to give 20 to 40 colonies perplate.To determine relative antibiotic activity of colonies,

a seed layer of B. subtilis was poured onto the platesafter 2 days. Zones of inhibition which developed werecompared, the relative sizes of the colonies being takeninto account, and yeast-glucose slants were streakedfrom colonies showing the highest activities. Theseslants were used to inoculate fermentation flasks.

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RESULTS

Morphological Variablity Upon Serial Transfer

The original culture of M366P1 on yeast-glucoseagar had a medium-brown vegetative mycelium, shortgray aerial hyphae, and white spores which developeda buff color with age. At 48 hours the growth was

layer and a decrease in exuded droplets were noted.No sector or definite decrease in sporulation was seenuntil transfer 58, which had fewer spores at the top ofthe slant. The area and degree of sparse sporulationincreased gradually upon subsequent transfer, untilin transfer 71 there appeared a heaped-up mealygrowth at the base, with web-like ridges of spores

Original

TrGnsfer 60 Transfer 70

Tran-sfer so ns er.

FIa. 1. Strain M366P1 on yeast-glucose agar plates: original culture and transfers 60, 70, 80, and 90.

uniformly covered with a thick, wrinkled spore layer,and a soluble brown pigment had diffused into thesubstratum. Exuded droplets were scattered over thestreaks and the base of the slants.This strain was transferred on yeast-glucose agar

slants every two days for 100 times. The morphologyappeared the same through the 25th (J) slant, afterwhich an increase in drying and cracking of the spore

over the rest of the slant. This heaped-up growthincreased and became more moist until transfer 85(+), when it began to decrease until the last culturesin the series were completely asporogenous or containeda few web-like ridges of spores. The mealy aerial growthwas creamy but still developed the buff color on aging.The thickness and color of the vegetative myceliumand pigmentation of the medium decreased con-

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DEGENERATION AND REGENERATION OF S. GRISEUS

siderably. Degenerated cultures (those after appearanceof mealy growth) did not change significantly in rateof growth.The same progression of changes was revealed when

inocula from the serial transfers were streaked out onplates of yeast-glucose agar (figure 1). Masses ofmealy growth and thin ridges of spores were scatteredover the streaks. When plates containing degeneratedcultures were stored at room temperature for 2 weeks,areas of normal sporulation developed at the edges ofstreaks made from transfers 71-77.

Microscopically, no difference was seen until thecultures showed sparse sporulation. Egg albuminimpression slides of asporogenous areas had both fewerspores and shorter chains. In the degenerated cultures,one-fourth to three-fourths of the spores or spore-likeunits were short ovoid and cylindrical rods the length

was started and carried to the 68th transfer. Thisseries showed essentially the same pattern of mor-phological degeneration, with pronounced decrease insporulation. Transfer 68 had only thin patches of thenormal type of sporulation scattered over the vegeta-tive mycelium.To see whether the pattern of degeneration was

characteristic of the strain or of the medium, maltosesporulation agar was inoculated from transfer 20 ofthe first series on yeast-glucose, and 45 serial transferswere made. On this medium, a pink color developedon the spore layer. Except for a wide variation inintensity of the pink pigment and in amount of exudeddroplets, M366P1 remained morphologically stable onthis medium.

Shorter series were run with strains W4-2, C-131,C-57, C, and V. Five transfers of each strain were

TABLE 1. Degeneration of Streptomyces griseus M866P1 during serial transfer

A. FIRST SERIES ON YEAST-GLUCOSE AGAR B. SECOND SERIES ON YEAST-GLUCOSE AGAR C. MALTOSE SPORULATION AGAR

Transfer | Day of peak| Peak potency pDrop in Transfer Peak potency Drop in Transfer Peak potency potenpcytTranserDa of pak lelL poency potency potency Droptincy

u/m u/mi%/ % u/Mi %

original 5 123 original 5312 5 68 45 10 4924 5 51 59 20 48 20 4936 3 57 54 30 101 30 27 4548 3 27 79 40 107 40 15 6960 3 45 64 45 30 4366 3-6* 47 62 50 27 49 50 13 7372 3-6* 10 92 55 11 79 55 8 8478 3-6* 4 97 60 10 8084 3-6* <1 >9990 3-6* 2 9896 3-6* <1 >99100 3-6* <1 >99

* Potencies at nearly same level, 3-6 days.t Drop in potency from transfer 20.

of two to three normal spores and slightly wider.These rods were not in chains. The spores of normalsize which were present appeared singly or in shortchains under ten.The inoculum for the serial transfers had always

been taken from the middle of the slant, and so de-creasing sporulation in that area could have hastenedthe degeneration. Since in the degeneration patternthe base was the last area to decrease in sporulation,inoculum from it should test this possibility. A sub-series was started from the base of transfer 36 (whichhad a normal spore layer over the entire surface),and inoculum for subsequent samples was also takenfrom the base. Nevertheless, this subseries showedthe same pattern of degeneration, and the mealysporulation appeared only four transfers later than inthe original series.To test whether the degeneration of M366P1 on

yeast-glucose agar was reproducible, a second series

made on yeast-glucose agar to determine whetherPotency dropped during only a few transfers, such asmight be made inadvertently in work with S. griseu8.All strains sporulated well and did not change mor-phologically. Forty-seven transfers were made onmaltose sporulation agar. Each strain showed a greatvariation in shade and intensity of the pink to pinkish-brown color on the spore layer, and slight variationsin amount of exudation and in surface wrinkling.Otherwise, all strains except W4-2 sporulated well andremained morphologically stable. W4-2 showed anoticeable drying and cracking of the spore layer afterabout 25 transfers.

Stability of Antibiotic Production Upon Serial TransferTable 1(A) shows a streptomycin assay of the first

series of M366P1. Similar results were obtained in otherassays made at different intervals in the degeneration,although tde actual values of antibiotic potencies and

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percentage decrease naturally varied somewhat withdifferent fermentations. In some assays, decreases asgreat as 87 per cent (from 45 to 79 per cent in theassay shown) were obtained before diminished sporu-lation or any other major morphological change wasapparent. All morphologically degenerated cultures hadless than 10 per cent of their original potency, andfinally practically no activity.

Slight flask to flask differences in pH occurred, butno correlation could be made between pH and progres-

TABLE 2. Streptomyces griseus strains V, C-181, and W4-* onyeast-glucose and maltose agars

STRAIN TRANSFER PEAK POTENCY

Yeast-glucose agar

u/ml

V 1 1493 1085 49

C-131 1 293 175 12

Maltose agar

V 1 6310 14520 10130 15540 90

C-131 1 335 1310 1120 830 840 7

W4-2 1 335 2010 420 330 340 2

sion from early to late transfers. However, beginningwith transfer 36 (Table 1(A), peaks in potency oc-

curred earlier and therefore at lower pH values, in therange of pH 7.8 to 8.0 instead of pH 8.4 to 8.5. Macro-scopically, it could be seen that fragmentation andlysis were taking place earlier in flasks inoculated fromtransfers 60 to 100. Microscopically, differentialstaining of hyphae, degree of fragmentation, and sporeformation were greater with these later transfers ateach sampling period.A few of the ten subcultures made from the M366P1

stock (see Materials and Methods) varied in potencyfrom the range of the others, 120 to 160 units/ml

under our conditions. Inadvertently, a culture laterfound to have a potency of only 50 to 80 units/mlwas used to inoculate the second series of M366P1;thus, the first transfer in this series assayed only 53units/ml. However, an interesting result was obtained.As shown in table 1(B), antibiotic production for noapparent reason increased in transfers 30 and 40 (101and 107 units/ml) and then showed a large decrease.Transfer 55 had dropped 79 per cent from the potencyof the first transfer, compared with transfer 60 in thefirst degeneration series, which had dropped 64 percent.

It will be recalled that the M366P1 series on maltoseagar was started from transfer 20 in the first serieson yeast-glucose agar. The potency of the first transferon maltose was 49 units/ml, which is in the range ofthe 51 units/ml obtained for transfer 24 in the yeast-glucose series. Table 1(C) shows that antibiotic pro-duction decreased 45 per cent between the first andtenth transfers on the maltose medium, whereas it didnot drop between the corresponding transfers on theglucose medium, that is, transfer 24 and 36 (table1(A). Although the cultures never showed any mor-phological degeneration on maltose agar, antibioticproduction decreased more than on the glucose medium.Transfer 60 assayed 10 units/ml in the maltose seriesand 45 units/ml in the yeast-glucose series.The potencies of strains C, N, and C-57 were too

low under our conditions of fermentation to warrantmore than a study of morphological variation. Table 2gives the results of assays on V, C-131, and W4-2. Vshowed a 67 per cent decrease in potency during 5transfers on yeast-glucose agar. On maltose agar theinitial activity was less (63 units/ml), but the potencyof transfer 10 was 145 units/ml, almost the same as theinitial value on yeast-glucose, and the potency wasstill high at the 40th transfer. The decreased activityof transfer 40 may be the beginning of a general declinein antibiotic production, or may represent a temporarydecrease. Unlike V, strains W4-2 and C-131 were notstimulated during transfer on maltose agar.

Regeneration of Antibiotic PotenciesThree methods were tried to reverse the degenera-

tion in antibiotic production of M366P1 in the firstseries on yeast-glucose agar: transfer to maltose andstarch sporulation agars, growth in soil, and selectionof natural variants.

Sporulation agars. Although decreases in antibioticpotency were obtained before any change in sporula-tion was seen, the decrease to almost no activity coin-cided with cultures becoming asporogenous. Thus, itwas thought that antibiotic potencies might rise ifthese cultures were induced to sporulate well. De-generated cultures from transfers 75 and 80 wereserially cultivated on maltose sporulation agar (used

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successfully in this laboratory with some poorlysporulating Streptomyces). In the series starting withculture 75, sporulation increased with each transfer,until the twelfth appeared the same as the originalM366P1 strain on this medium. Twenty serial transferson maltose agar were made starting with culture 80,but although the normal type of sporulation reappeared,the spore layer remained thin with asporogenous

areas.The normal type of sporulation returned at the

first transfer of degenerated cultures 75, 80, 90, and100 to starch sporulation agar. A normal amount ofsporulation was present after 5 to 10 transfers. Starchtransfers 1, 5, 10 from each of the above four cultureswere assayed along with maltose transfers 1, 6, 12from culture 75 and 1, 10, 20 from culture 80. Allgave less than 10 units/ml except the maltose andstarch transfers from culture 75, which had potenciesof 14-25 units/ml. Since culture 72 in the degenerationseries assayed 10 units/ml and culture 78 4 units/ml,transfers on maltose and starch agars starting fromculture 75 showed a slight increase over those at thebeginning of mealy sporulation, but did not approachthe antibiotic production of the original culture.Asporogenous strain N was transferred 10 times on

both the maltose and starch agars, but the cultureswere completely asporogenous or contained only a

few ridges of spores. Several slants were assayed butnone showed any activity.

Soil. The original culture and every tenth transfer,except for 75 in place of 70, were inoculated into tubesof sterilized soil and allowed to grow. Transfer 75was included in order to have a culture from near thebeginning of degeneration. It will be recalled thatmealy growth first appeared in transfer 71. Sporulationin the soil was noted in all tubes except those inoculatedfrom transfers 75, 80, 90, and 100. After 2 weeks ofincubation at 30 C, transfers were made to yeast-glucose agar slants which were used to inoculatefermentation flasks. The morphology of all cultures on

these slants was the same as on corresponding serialtransfers, and all potencies were in the range of thoseof the corresponding serial transfers. Thus, growth insterile soil did not change the antibiotic-producingability of degenerated cultures or of those with normalmorphology.

Natural variants. To find natural variants whichwere higher producers of streptomycin, inocula fromthe original culture and every tenth transfer (exceptfor 75 in place of 70) were plated out on yeast-glucoseagar as described in Materials and Methods. A spore

inoculum was obtained for all cultures but 80, 90, and100. Since mycelium and spore-like units in the lattercultures could not be separated by centrifugation, a

biotic activity of colonies was determined by thecolony assay method.

All of the largest zones were around colonies withthick spore caps; however, many colonies with goodsporulation had small or no zones. All asporogenousand mealy colonies had small or no zones. Both normaland mealy spore caps appeared on plates from transfer75, but no colonies from transfers 80, 90, and 100 hadthe normal type of sporulation. From a total of 217plates in 5 platings, with an average of about 30

TABLE 3. Selection of natural variants of Streptomyces griseus

COMPARISON WITH TRANSFERS

TRANSFER PLATES PEAK POTENCY OF IN DEGENERATION SERIESTRANSFER PlATESXSLE.CTED COILONIES ______-____Transfer Potency

u/ml u/mi

original 4 125 (2) original 123105

10 4 none assayed

20 4 160* 12 68110* 24 518372

30 24 105* 36 5796*89*83*

31-69 (5)

40 4 2823

50 24 25-55 (6) 48 27

60 54 105* (2) 60 4596*

75 54 105* 72 1096* (3) 78 474*

80, 90, 100 15 each none assayed

* At least 40 per cent higher than potency range of parentculture.

colonies per plate, 32 colonies were tested for potencyin fermentation flasks. Results are reported in table 3.Occasional difficulty in judging the relation of zonesize to colony size may explain the selection of somecolonies which had low potencies.

DISCUSSIONThe appearance of the degenerated cultures cor-

responds to the description of genus Nocardia in the6th edition of Bergey's Manual of Determinative Bac-teriology (Breed et al., 1948). "The colonies of nocardiashave a paste-like or mealy consistency.... Typicalnocardias never form an aerial mycelium, but there are

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niixed inoculum was taken off their surfaces. Anti-

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cultures whose colonies are covered with a thin coatingof short aerial hyphae, which break up into cylindricaloidiospores." Other reports of transitions betweenstreptomycetes and nocardias appear in the literature.In an examination of 1,298 freshly isolated cultures ofStreptomyces, Jones (1949) stated that one-fifth fluctu-ated in production of aerial mycelia, and that assign-ment of the fluctuating group to Streptomyces or

Nocardia would be entirely arbitrary. Schatz andWaksman (1945) found that sporogenous areas oftendeveloped on old stationary cultures of inactive non-

sporulating strains of S. griseus, and described thesestrains as intermediate. Appleby (1948) reported an

asporogenous variant of S. griseus which could bepropagated on a variety of media without reversion.

In the present study, asporogenous cultures were

apparently stable on some media but would revert on

others. The degenerated asporogenous variant ofM366P1 remained stable on yeast-glucose and maltosemedia, but was easily transformed to the sporogenousculture on starch medium. N remained asporogenouson all three media. Areas of normal sporulation de-veloped at the edges of streaks made from degeneratedtransfers 71-77. These may correspond to Waksman's"intermediate" asporogenous variants. Which of thedegenerated cultures should be classified as Strepto-myces and which as Nocardia? According to the classifi-cation in the 6th edition of Bergey's Manual, the transi-tion is not only between genera, but between twofamilies, Streptomycetaceae and Actinomycetaceae.

Carvajal (1946), Waksman and Harris (1949), andDulaney et al., (1949) could find no definite correlationbetween morphological or cultural characteristics andstreptomycin production. The only correlation betweenmorphology and antibiotic production in the presentstudy was that asporogenous cultures or those withthe mealy type of sporulation had potencies of 10units/ml or less. Many colonies with good spore capswere poor producers or inactive, and serial transferswhich could not be distinguished morphologicallyfrom the original cultures had dropped significantly in

antibiotic production.Waksman (1949) has reported that asporogenous

inactive cultures of S. griseus which revert to thesporulating strain will produce streptomycin. In thepresent work, antibiotic-producing ability was in-creased slightly with reappearance of sporulation ina culture near the beginning of degeneration (transfer75), but not in cultures farther along in the series(transfers 80, 90, 100).

Transfers in all the series showed a marked variationin pigmentation on the spore layer and in amount ofexudation. Besides this normal variation in deliques-cence, a drying and cracking of the spore layer withdecrease in exuded droplets was always noticed before

morphologically on maltose agar than on yeast-glucose,but potencies decreased faster on maltose. This indi-cates that the optimum artificial medium for preservinghigh potencies in S. griseus is not necessarily themedium which gives the most stable morphology.

In fermentation shake flasks, degenerated culturesshowed earlier fragmentation and lysis and producedmore spores. Similar results have been reported byother workers. Schatz and Waksman (1945) foundthat antibiotically active strains took about twice aslong to undergo lysis as did asporogenous inactivestrains. Carvajal (1947) stated that a non-streptomycin-producing strain of S. griseus showed better sporulationin,shake flasks than any of the streptomycin-producingstrains did under the same conditions. He did notsay whether the parent culture was sporogenous orasporogenous.Although Jones (1946) and Erikson (1947) found

that sporulation in some streptomycetes was restoredby growing in sterilized soil, this method was notsuccessful in the present study. Growing the culturesin soil preserved both morphology (including thedegenerate type) and antibiotic-producing ability, butdid not increase the latter. Natural variants with higherstreptomycin-producing ability were selected fromsporogenous and poorly sporulating cultures and fromone near the beginning of the degeneration (transfer75), but not from cultures farther along in the de-generation (transfers 80, 90, 100). These variants wererelatively few, and it may be concluded that a largenumber of samples would have to be plated out toobtain superior strains by this method.Waksman (1950) has stated that the ability to form

streptomycin may be considered a strain propertyrather than a species characteristic like the formationof penicillin by the Penicillium notaum-P. chry8ogenumgroup of fungi. Thus, it is not surprising that anti-biotic production by P. chrysogenum (Reese et at.,1949) should be more stable to serial transfer. Thepresent study has shown that the antibiotic-producingability of S. griseu is not stable upon transfer onartificial media, and that the antibiotic production ofa culture cannot be judged by its morphologicalappearance.

SUMMARYDuring 100 serial transfers on yeast-glucose agar

strain M366P1 of Streptomyces grieus showed a pro-gressive degeneration into cultures morphologicallytypical of Nocardia. After 58 transfers, the culturesfirst decreased in sporulation, then developed a mealygrowth, and finally became asporogenous. Shorterseries of transfers were made of six other strains of S.griseUs.

Significant decreases in potency occurred beforedecreased sporulation or any other morphological

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decreased sporulation. M366P1 was more stable

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change was apparent. The only correlation betweenmorphology and antibiotic production was that asporog-enous cultures or those with a degenerate type ofsporulation gave yields of 10 units/ml or less. Insubmerged fermentations, degenerated cultures showedearlier lysis and produced more fragmentation spores.Normal aerial sporulation was restored by trans-

ferring degenerated cultures on maltose and starchagars. This increased antibiotic production slightly ina culture near the beginning of degeneration, but notin cultures farther along in the transition. Growth insterilized soil preserved morphology (including thedegenerate type) and antibiotic-producing ability,but did not increase the latter.

Natural variants with higher potencies were selectedfrom cultures with normal morphology and from onenear the beginning of degeneration, but not fromcultures well along in the transition. However, thesesuperior variants were relatively few.

REFERENCESAPPLEBY, J. C. 1948 An asporogenous variant of Strepto-

myces griseus. J. Gen. Microbiol., 2, 80-82.BREED, R. S., MURRAY, E. D. G., AND HITCHENS, A. P. 1948

Bergey's Manual of Determinative Bacteriology. 6th Ed.The Williams & Wilkins Co., Baltimore, Md.

CARVAJAL, F. 1946 Biological strains of Streptomyces griseus.Mycologia, 38, 596-607.

CARVAJAL, F. 1947 The production of spores in submergedcultures by some Streptomyces., Mycologia, 39, 426-440.

DULANEY, E. L., RUGER, M., AND HLAVAC, C. 1949 Observa-tions on Streptomyces griseus. IV. Induced mutationand strain selection. Mycologia, 41, 388-397.

ERIKSON, DAGNY 1947 Differentiation of the vegetative andsporogenous phases of actinomycetes. 2. Factors affect-ing the development of the aerial mycelium. J. Gen.Microbiol., 1, 45-52.

JONES, K. L. 1946 Further notes on variation in certainsaprophytic actinomycetes. J. Bacteriol., 51, 211-216.

JONES, K. L. 1949 Fresh isolates of actinomycetes in whichthe presence of sporogenous aerial mycelia is a fluctuatingcharacteristic. J. Bacteriol., 57, 141-145.

Loo, V. H., SKELL, P. S., THORNBERRY, H. H., EHRLICH, J.,MCGUIRE, J. M., SAVAGE, G. M., AND SYLVESTER, J. C.1945 Assay of streptomycin by the paper disc-platemethod. J. Bacteriol., 50, 701-710.

REESE, E., SANDERSON, K., WOODWARD, R., AND EISENBERG,G. M. 1949 Variation and mutation in Penicillium chrys-ogenum, Wis. Q176. J. Bacteriol., 57, 15-21.

SCHATZ, A., AND WAKSMAN, S. A. 1945 Strain specificity andproduction of antibiotic substances. IV. Variationsamong actinomycetes with special reference to Actinomycesgriseus. Proc. Nat. Acad. Sci., U. S. 31, 129-137.

WAKSMAN, S. A. 1949 Streptomycin. The Williams &Wilkins Co., Baltimore, Md.

WAKSMAN, S. A. 1950 The Actinomycetes. Chronica Bo-tanica Co., Waltham, Mass.

WAKSMAN, S. A., AND HARRIS, D. A. 1949 Streptomycin-producing capacity of different strains of Streptomycesgriseus. Procr. Soc. Exptl. Biol. Med., 71, 232-235.

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