System of Color Wheels for Streptomycete TaxonomyH. D. TRESNER AND E. J. BACKUS

Biochemical Research Section, Lederle Labor-atories Division, American Cyanamid Company, Pearl River, New York;

Received for publication 13 February 1963

ABSTRACT

TRESNER, H. D. (Lederle Laboratories Division,American Cyanamid Co., Pearl River, N.Y.) AND E. J.BACKUS. System of color wheels for streptomycete taxon-omy. Appl. MVicrobiol. 11:335-338. 1963.-In the sundrysystems of streptomycete taxonomy, color of the sporu-lating aerial mycelium is frequently employed as a system-atic criterion. Color series, each containing species ofsimilar spore colors, are generally erected; however, therange of colors encompassed within a series is often notclearly delineated by the usual word description. There-fore, a system is proposed in which the color content ofeach series is more accurately defined by means of colortabs. Seven spore-color series are recognized (i.e., red,gray, yellow, blue, green, violet, and white), each ofwhich is represented by a color wheel that displays therange of colors included therein. By comparing sporecolors with the color wheels, unclassified isolates canreadily be assigned to appropriate color groups.

The color of the sporulating aerial mycelium of thestreptomycetes is a feature that has been used extensivelyfor taxonomic purposes, and various systems of colorgroupings have been proposed. Casual inspection of thesesystems suggests that there is considerable divergence ofopinion among investigators regarding the content of thegroupings recognized. If, however, closer inspection ismade, some of the differences prove to be more apparentthan real.

In some of the more recent classification schemes, inwhich color of aerial mycelium plays an integral role, acertain continuity does seem to exist between the commoncolor categories recognized. In Table 1, five color systemsof varying complexities are compared. An attempt hasbeen made to group together the similar common colornames in each system and to arrange them in the sameorder. When this is done, either five or six general colorgroups per system are formed: i.e., those having white,yellow, red, gray, and blue or green, or both, as miajorcomponents. It will be noted that, in the Pridham, Hes-seltine, and Benedict (1958) and the Ettlinger, Corbaz,and Hutter (1958) systems, each general color group thatis recognized is represented by a single color series. How-ever, in the other three systems the common color groupsare mostly subdivided into multiple series that are formed

on the basis of different combinations of aerial mycelial(en masse spores) colors with different colors of vegetativemycelium. When the larger color categories are dividedinto the various series, the main intent, of course, is tosystematically separate the streptomycetes into as manysmaller parcels. Ideally, the descriptive color terminologyemployed with these series should, at the same time,effectively define the range of spore colors exhibited bythe organisms contained therein. Unfortunately, this isnot always accomplished.

In practice, attempts at written communication ofcolor terminology between individuals often prove to bevery confusing. Color designations meaningful to one maysuggest something quite different to another. Hence, it isnot surprising that, in the many existing taxonomicsystems, different color terms are used to describe colorseries which are undoubtedly the same or comparable.Examples of this may be observed in Table 1. It wouldseem, then, that, even though the various color systemsmay appear dissimilar, in reality many of the differencesare only superficial and primarily a matter of semantics.The use of a uniform descriptive color terminology,

perhaps employing a single color code, could be useful inattaining better universal agreement on color valuesamong streptomycete taxonomists. Toward achievementof this objective, we are proposing a new system thatrecognizes seven spore-color series, each represented by acolor wheel composed of selected color chips from theColor Harmony Manual (Jacobson, Granville, and Foss,1948) which set forth the range of colors encompassed byeach individual series.

MATERIALS AND METHODS

Color may play an important role in the classificationof the streptomycetes, providing that proper use is madeof this criterion. When the sporulating aerial mycelium ofmature cultures is observed under suitable lighting con-ditions, it will be noted that each culture exhibits acharacteristic color. Although the specific shade may varyslightly from time to time depending upon the depth ofthe sporulation layer, intensity of underlying pigmenta-tion, age of culture, type of medium, etc., nevertheless,the culture can be reliably assigned to a general color,e.g., grayish, bluish, greenish, etc. When mass collectionsof streptomycetes are classified according to this method,it is observed that all well-sporulated cultures generallyfall readily into a relatively few such color groups. It has

335

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been our experience that seven color series accommodateall the streptomycetes observed to date.

It is virtually impossible to apply color terminology tothese series that will define with precision for everyonethe range of color shades represented in each group.

TABLE 2. Color Harmony Manual code and common-namledesignations for the hues included in the seven

spore-color series

CHMI code CHM hues CHM code CHM hues

Colors in Red (R)series

Lt. Ivory; EggshellLt. Melon YellowLt. ApricotNude Tan; RoseBeige

Peach TanCork TanLt. Fawn; RoseBeige

RosewoodPussywillow GrayPowder RoseDusty PeachBisque; Lt. RoseBeige

Bisque; Lt. BeigeNo Name (near

grays)Baby Pink; PalePink

Flesh Pink; PalePeach; Shell Pink;Tearose

Pearl Pink; Shell

Colors in Blue (B)series

Lt. AquaAqua GrayDusty Aqua GreenAqua GrayBayberry GrayMed. Blue SpruceJade GrayBlue Spruce

Colors in Yellow (Y)series

IvoryPearl; Shell TintYellow TintParchmentPastel YellowPastel YellowBamboo; Buff;Straw; Wheat

No Name (neargrays)

Putty; GriegePuttyParchment

2 dc2 ge

3 ge

4 ig4 li3 li3 ig

2 ih3 ih5 ih

7 ih7 fe5 fe

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10 ec

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Colors in Gray (GY)series

Natural; StringCovert Tan; GriegeBeige; CamelFawnBeaverBeaverBeige Brown; MistBrown

Dark Covert GrayBeige Gray; MouseLead Gray;Shadow Grav

Taupe GrayAshesAshesGray (gray scale)Gray (gray scale)No Name (gray

scale)Covert GraySilver Gray

Colors in Green(GN) series

Lt. Olive GrayOlive GrayOlive GrayLt. Olive DrabLt. Olive DrabMistletoe GraySage GrayDk. Reseda Green;Sage Green

Mistletoe GreenIvy

Colors in Violet (V)series

Pale LilacOrchid MistWistariaOrchidLilac

Colors in White (W)series

WhiteOyster WhiteAlabaster Tint

Therefore, we find it preferable to apply a simple colordesignation to each recognizable group and then definethe content of each group by more accurate and graphicmeans.To accomplish this, the color of sporulation of several

hundreds of streptomycetes, both described species andunclassified soil isolates, was matched under north-windowdaylight with color tabs of the Color Harmony Manual.We have observed that this manual provides an excellentcolor guide for matching the subtle, pastel shades ex-pressed in the spores of the streptomycetes. In almost allinstances, we were able to find color tabs which closelyapproximated the spore colors, of the organisms studied.Then, by selecting these particular chips and arrangingthem into their respective color groups according to certainunderlying color similarities, the range of colors coveredand the limits of the seven series were defined. Since theconcept of these color series was already preconceived,but was lacking in clear-cut definition, arrangement of thechips into the different groups did not entirely dictate theoutlines of each series. It did, however, help clarify thereasons spore-color groups suggest themselves when oneinspects masses of isolates. For example, certain culturesall had in common some shade of gray, whether it belight gray, dark gray, brownish-gray, olive-gray, etc.Still others had some shade of red in combination withother colors, to give a wide variety of pinkish to pinkish-cinnamon values, but all containing the basic red element.Other natural series had yellow, green, blue, violet, orwhite as the underlying color in common.

For the sake of convenience and practicality in makinguse of the different color groupings, the Color Harmony

FIG. 1. Wheel arrangement of the Color Harmony Manual chips as

nised in the various color series.

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V'OL. 11) 1963 337

TRESNER AND BACKUS

Manual chips in each series were arranged into colorwheels (Fig. 1). An attempt was made to place the chipsaround the wheels so that they formed a continuous andharmonious spectrum within each series. However, thiswas not always possible, since many hues, needed to form acomplete continuum, were not represented by any of thestreptomycetes examined. It is possible that some of themissing hues could eventually be found were enoughorganisms studied. This, of course, is of little consequence,since, in using the system as it is designed, the objectiveis not necessarily to match with precision the exact shadeof an organism, although this is frequently possible; rather,it is to determine simply to which general color series itbelongs. Even though the spore color of a culture maybe intermediate between those tabs represented in a colorwheel, this becomes obvious to the observer upon inspec-tion, and does not cause any particular difficulty in estab-lishing the color series relationship.

RESULTS AND DISCUSSION

As i-idicated earlier, definition of each color series byall-inclusive descriptive terms is impractical and unneces-sary; therefore, we have designated them simply as thegray (GY), red (R), yellow (Y), blue (B), green (GN),violet (V), and white (W) series. In Table 2, the ColorHarmony Manual values of the various tabs in each ofthese color series are given in the order they appear inthe color wheels. Both code designations and commonnames are provided for each chip.

In making application of the color wheel system fortaxonomic purposes, certain procedures need to be ob-served in order to use it most effectively. For example,comparison of color tabs with cultures should be made innorth-window daylight, preferably on a bright day orunder comparable artificial lighting conditions. (A satis-factory substitute is provided by the Macbeth Daylight-ing Lablite model BBX-526 with Color Matching Booth,Macbeth Daylighting Corp., Newburgh, N.Y.) Only thedull or nonreflective surface of the Color Harmony Manualchips should be used. It may even be advantageous toremove a tab from the wheel for careful comparisons.

Cultures being studied should be mature (about 2 to 3weeks old), except in instances where spore masses undergobreakdown and become hygroscopic and darkened withage; spore color of these organisms should be measuredprior to such changes. The presence of spore masses,sufficiently heavy to characterize the color of the aerialgrowth, should always be ascertained before attemptingto make a color-tab comparison. The measurement of thecolor of nonsporing aerial mycelium is of no value forpresent purposes, since it is subject to considerable varia-tion depending upon cultural and nutritional factors.

Cultures should be observed on several media beforemaking spore-color determinations. In this way, one isable to detect and discount the interfering influencesexerted by the color of underlying substrate thallus, pig-ments, or medium. The color measurement is best made on

clear media that provide good sporulation but do notsupport abundant pigment formation by the organisms.As a result of the wide array of colors and shades ex-

hibited by the spores in the many species of streptomyces,a clear-cut separation of color series is not possible. Forthis reason, there are a few locations on certain of thecolor wheels in which an individual tab will be near incolor to one in another series. For example, the followingcombinations of Color Harmony Manual chips havesimilarities which might cause some confusion in usingthe system: 112 ge (G\N series) and 112 ec (Y series);2 ca (R series) and 2 db or 2 ba (Y series); 4 ge (R series)and 4 ig (GY series); and 3 ec (R series) and 3 ge (GYseries). However, this need not be a serious defect in thesystem as long as provisions are made for such recognizedsimilarities. A simple solution to this problem, of course,is to classify the relatively few species which fall into suchquestionable positions into both of the color series in-volved. Then, by a device commonly used in taxonomickeys, both pathways followed to the species level can bedesigned to converge to a common position in the key.

It has been our experience that, when diligence is prac-ticed in observing and matching colors under proper light-ing conditions, most streptomycetes can be readily cate-gorized according to the color series we recognize. Throughthe use of a system, such as proposed here, in which thecolor series are carefully defined, sporulation color thenibecomes a very useful primary taxonomic implement inthe classification of the streptomycetes.

ACKNOWLEDGMENTSThe foregoing color wheel system was presented at the

International Subcommittee on Taxonomy of the Actino-mycetales Workshop held at the Department of Bacteriol-ogy and Immunology, MIcGill University, Montreal,Canada, 18 August 1962. The authors wish to expresstheir appreciation for the conistructive comments andsuggestions about the system offered by those who at-tended the session.

LITERATURE CITEDBALDACCI, E. 1959. Development in the classification of actinomy-

cetes. Giorn. Microbiol. 6:10-27.ETTLINGER, L., R. CORBAZ, AND R. HtTTER. 1958. Zur systematik

der actinomyceten. 4. Eine Arteinteilung der gattung Strep-tomyces Waksman et Henrici. Arch. Mikrobiol. 31:326-358.

GAUZE, G. F., T. P. PREOBRAZHENSKAYA, E. S. KUDRINA, N. 0.BLINOV, I. D. RYABOVA, AND M. A. SVESHNIKOVA. 1957. Prob-lems of classification of actinomycete-antagonists. Institutefor Research of New Antibiotics, Academy of MedicalSciences, National Press of Medical Literature, Medzig, Mos-cow.

JACOBSON, E., W. C. GRANVILLE, AND C. E. Foss. 1948. Colorharmony manual, 3rd ed. Container Corporation of America,Chicago.

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

WAKSMAN, S. A. 1961. Classification, identification and descriptionof genera and species. The actinomycetes, vol. 2. The Williams& Wilkins Co., Baltimore.

338 APPL. I'XIICROBIOL.