Taxonomy by Carbon Replication · Taxonomy by Carbon Replication I. An Examination of Streptomyces hygroscopicus ALMA DIETZ AND JOHN MATHEWS Research Laboratories, The Upjohn Coompany,

Taxonomy by Carbon Replication

I. An Examination of Streptomyces hygroscopicus

ALMA DIETZ AND JOHN MATHEWS

Research Laboratories, The Upjohn Coompany, Kalamazoo, Michigan

Received for publication December 20, 1961

ABSTRACT

DIETZ, ALMA (The Upjohn Company, Kalamazoo,AIich.) AND JOHN MATHEWS. Taxonomy by carbonreplication. I. An examination of Streptomyces hygro-scopicus. Appl. Microbiol. 10:258-263. 1962.-Pre-shadowed carbon replication of spore surfaces (carbonrepligraphy) provides a new technique for the characteri-zation of streptomycetes. Carbon repligraphs of fivemembers of the Streptomyces hygroscopicus complex showtwo distinct types. Type I shows a nonsegmented sporestructure with an extremely wrinkled surface. Type IIhas a segmented spore chain with detailed surface structureresembling a basket weave.

1'revious attempts to characterize actinomyeetes havebeen based primarily on color (Dietz, 1954), carbonutilization (Pridham and Gottlieb, 1948), and morphologyof the sporophore (Pridham, Hesseltine, and Benedict,1958). Direct examination of spores by the electronmicroscope provides one more criterion for characteriza-tioIn (Baldacci and Grein, 1955; Pridham, 1959; Tresner,Davies, and Backus, 1961). This method produces only asilhouette of the spore and fails to adequately differentiatethe various species.

Information obtained by examination of preshadowedcarbon replicas of spores shows rich surface detail whichmakes possible a critical comparison of otherwise closelyrelated streptomycetes. For simplicity, we will refer toelectron micrographs of preshadowed carbon replicas ascarbon repligraphs. Carbon repligraphy is presented as anew taxonomic method.

M1ATERIALS AND METHODS

1F,ive cultures showing the fundamental characteristicsof Streptomyces hygroscopicus (Jensen), Waksman andHenrici (Jensen, 1931; Tresner and Backus, 1956; Waks-man, 1961) were chosen. These organisms were: Strepto-niyces endus, NRRL 2339; S. hygroscopicus, Lilly M5-13184; S. hygroscopicus var. decoyicus, NRRL 2666; S.hygroscopicus var. angustmyceticus, NRRL B-2347; andStreptomyces rutgersensis var. castelarense, Cepa 41-18.

Macroscopic comparison. The cultures were grown underthe following conditions:

1) Tryptone-yeast extract broth (tryptone, 0.5 %;

yeast extract, 0.3 %; distilled water) dispensed in 100-mlamounts in 500-ml Erlenmeyer flasks. F'lasks autoclaved15 min at 15 psi (120 C).

2) Sterile flasks seeded from soil stocks of the culturescited and incubated 48 hr at 28 C on a reciprocal shaker.

3) Growth blended 1 min at slow speed.4) Blended inoculum, 0.2 ml, seeded on surface of 6

agar media1 in duplicate tubes.5) After 7 days of incubation at 28 C, tubes photo-

graphed on Ektacolor,2 type S,3 to show obverse andreverse of each culture.

Direct examination.4 For direct examination, carbon-covered grids were touched to the surface of 7-day aerialgrowth on Bennett agar seeded at the same time as thetubes. A large number of spores became attached to thecarbon surface and were observed with the electronmicroscope.

Carbon repligraphy.4 Preshadowed carbon replicas wereprepared in the following manner:

1) Touch surface of 7-day aerial growth with flat surfaceof small cover slip.

2) Place cover slip in vacuum evaporator and depositchromium film at an angle of 20 from the horizontal.

3) Deposit carbon film normal to the surface.4) Remove cover slip from evaporator and scratch

carbon-chromium film into 2-mm squares.

1 All constituents are given per liter of distilled water. Whereindicated, pH adjustment is made before addition of agar.

1) Bennett's agar: yeast extract, 1 g; beef extract, 1 g; N-Zamine A, 2 g; glucose, 10 g; (pH 7.0); agar, 15 g.

2) Czapek's sucrose agar: NaN(3, 2 g; K2HP04, 1 g; MgSO4-7H20, 0.5 g; KCl, 0.5 g; FeS04-7H20, trace; sucrose, 30 g; (pH6.6); agar, 15 g.

3) Maltose tryptone agar: maltose, 10 g; tryptone, 5 g; K2HP04,0.5 g; NaCl, 0.5 g; FeSO4*7H20, trace; agar, 15 g.

4) Peptone iron agar: peptone, 15 g; proteose-peptone, 5 g;ferric ammonium citrate, 0.5 g; K2HP04, 1 g; Na2S203, 0.08 g;agar, 15 g.

5) Tyrosine agar: i.-tyrosine, 1 g; agar, 15 g.6) Casein starch agar: Na-caseinate, 2 g; soluble starch, 1 g;

K2HP04, 0.2 g; MgSO4-7H20, 0.2 g; FeSO4 7H20, trace; agar,15 g.

2 Eastman Kodak Company, Rochester, N. Y.3 We are indebted to Norman Drake, The Upjohn Company,

for the color prints.4 Seven-day cultures of each organism were examined at least

five different times.

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TAXONOMY BY CARBON REPLICATION

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FIG. 1. Streptomyces endus, whole spore mount.FIG. 2. S. endus, carbon repligraph of spore.FIG. 3. Streptomyces hygroscopicus, whole spore mount.FIG. 4. S. hygroscopicus, carbon repligraph of spore.Each index mark equals 1 ,u.

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FIG. 5. Streptomyces hygroscopicus var. decoyicus, whole spore mount.FIG. 6. S. hygroscopicus var. decoyicus, carbon repligraph of spore.

FIG. 7. Streptomyces hygroscopicus var. angustmyceticus, whole spore mount.FIG. 8. S. hygroscopicus var. angustmyceticus, carbon repligraph of spore.

Each index mark equals 1 ,u.

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TAXONOMY BY CARBON REPLICATION

FIG. 9. Streptomyces rutgersensis var. castelarense, whole spore mount.FIG. 10. S. rutgersensis var. castelarense, carbon repligraph of spore.FIG. 11. Streptomyces hygroscopicus var. angustmyceticus, carbon repligraph at higher magnification.FIG. 12. Streptomyces hygroscopicus var. decoyicus, carbon repligraph at higher magnification.Each index mark equals 1 ,u.

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FIG. 13. Streptomyces endus.FIG. 14. Streptomyces hygroscopicus.FIG. 15. Streptomyces rutgersensis var. castelarense.FIG. 16. Streptomyces hygroscopicus var. decoyicus.FIG. 17. Streptomyces hygroscopicus var. angustmyceticus.

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TAXONOMIY BY CARBON REPLICATION

5) Immerse cover slip in aqueous 25 %o KOH overnightto dissolve spores.

6) Tease carbon films from cover slip and, using a2-cm square piece of 200-mesh bronze screen, transferfilms to surface of distilled water.

7) Transfer carbon films to surface of 0.2 N HCl fol-lowed by two more distilled water rinses.

8) Pick up carbon films on 200-mesh grids and air dry.These whole spore mounts and replicas were examined

in an Akashi Tronscope5 and photographed on Kodakmedium lantern slide plates at an electronic magnificationof 10,000X.

RESULTS AND DISCUSSION

Ektacolor prints, Fig. 13 to 17, indicate the culturalsimilarity of the five chosen streptomycetes. All have acomparable color pattern with two exceptions: S. rutger-sensis var. castelarense, Fig. 15, produced an intense redpigment on no. 5 medium; and S. hygroscopicus var.decoyicus, Fig. 16, and S. hygroscopicus var. angustmy-ceticus, Fig. 17, show more luxuriant growth on mediumno. 2. All five organisms had the distinctive hygroscopiccharacteristic and typical compact spiral growth associ-ated with the S. hygroscopicus complex.

S. endus, shown in Fig. 1 and 2, has a rough, apparentlynonsegmented, spore structure, Fig. 1. The carbon repli-graph, Fig. 2, shows the surface to be deeply wrinkled andnonsegmented.

Figures 3 and 4 show S. hygroscopicus. Figure 3 appearssimilar to Fig. 1. Figure 4, although showing a less rugosesurface, is comparable in general morphology to Fig. 2.

S. hygroscopicus var. decoyicus, shown in Fig. 5 and 6,has a segmented spore chain which appears relativelysmooth in lFig. 5. The carbon repligraph, Fig. 6, shows afinely contoured surface with the spores apparentlyencased in a loose sac. An enlargement of a portion of thisspore chain is seen in Fig. 12.

S. hygroscopicus var. angustmyceticus, Fig. 7 and 8, iscomparable with Fig. 5 and 6. The same type of sporechain is seen and the surface contour is similar althoughnot identical. Note the fine detail in the left center portionof Fig. 8. An enlargement of this area is seen in Fig. 11.

S. rutgersensis var. castelarense is seen in Fig. 9 and 10.Figure 9 shows similarity to Fig. 1 and 3 with a rough,nonsegmented structure. Figure 10 resembles Fig. 2 insurface detail.

5 Akaski Seisakusho, Ltd., Tokyo, Japan

It should be pointed out that the apparently nonseg-mented spore structures of S. endus, S. hygroscopicus var.decoyicus, and S. rutgersensis var. castelarense are mis-leading. Specimens partially dissolved with potassiumhydroxide show the presence of transverse septa dividingthe structure into individual spores. Older cultures showthe spiral spore structure breaking into individual spores.

Five organisms closely related on the basis of color andmorphology are divided into two types on the basis ofexamination of carbon repligraphs. S. endus, S. hygro-scopicus, and S. rutgersensis var. castelarense comprisetype I, a group of organisms having a nonsegmented,rugose spore structure. S. endus and S. rutgersensis var.castelarense appear identical and closely related to S.hygroscopicus, the type species.

S. hygroscopicus var. decoyicus and S. hygroscopicus var.angustmyceticus are similar on the basis of the segmented.finely contoured spores and comprise type II.We believe that the preshadowed carbon replica tech-

nique provides a powerful tool for the critical characteri-zation of the streptomycetes.

LITERATURE CITED*BALDACCI, E., AND A. GREIN. 1955. Esame della forma delle spore

di attinomiceti al microscopic electronico e loro valutazioneai fini di una classificazione. Giorn. microbiol., 1:28-34.

DIETZ, A. 1954. Ektachrome transparencies as aids in actinomyceteclassification. Ann. N. Y. Acad. Sci. 60:152-154.

JENSEN, H. L. 1931. Contribution to our knowledge of the Actino-mycetales. II. The definition and subdivision of genusActinomyces, with a preliminary account of Australian soilactinomycetes. Proc. Linnean Soc. N. S. Wales 56:345-370.

PRIDHAM, T. G. 1959. Retrospections on streptomycete taxonomy.Rev. latinoam. microbiol. Suppl. 3:1-22.

PRIDHAM, T. G., AND D. GOTTLIEB. 1948. The utilization of carboncompounds by some actinomycetales as an aid for speciesdetermination. J. Bacteriol. 56:107-114.

PRIDHAM, T. G., C. W. HESSELTINE, AND R. G. BENEDICT. 1958.A guide for the classification of streptomycetes according toselected groups. Placement of strains in morphological sec-tions. Appl. Microbiol. 6:52-79.

TRESNER, H. D., AND E. J. BACKUS. 1956. A broadened conceptof the characteristics of Streptomyces hygroscopiciis. Appl.Microbiol. 4:243-250.

TRESNER, H. D., E. C. DAVIES, AND E. J. BACKUS. 1961. Electronmicroscopy of streptomyces spore morphology and its rolein species differentiation. J. Bacteriol. 81:70-80.

WAKSMAN, S. A. 1961. The actinomycetes. vol. II. The Williams &Wilkins Co., Baltimore. 375 p.

* Since this manuscript was prepared, D. A. HoPWOOD AND

A. M. GLAUERT (J. Gen. Microbiol. 1961. 26:325-330) describe thefine structure of the spore surface of a single species, Streptomnycesviolaceoruber.

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