Vol. 46. No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1983. p. 925-9290099-2240/83/100925-05$02 .00/0Copyright © 1983, American Society for Microbiology

Novel Method for Selective Isolation of ActinomycetesC. F. HIRSCH* AND D. L. CHRISTENSEN

Merck Sharp & Dolitne Researclh Laboratories, Merck & Co., Inc., P.O. Box 2000, Rahway,News Jersey 07065

Received 17 March 1983/Accepted 12 July 1983

A new technique for the selective isolation of actinomycetes from natural mixedmicrobial populations is described. A nutrient agar medium was overlaid with a0.22- to 0.45-pm-pore cellulose ester membrane filter, and the surface of the filterwas inoculated. During incubation, the branched mycelia of the actinomycetespenetrated the filter pores to the underlying agar medium, whereas growth of non-actinomycete bacteria was restricted to the filter surface. The membrane filterwas removed, and the agar medium was reincubated to allow the development ofthe isolated actinomycete colonies. This procedure selects actinomycetes on thebasis of their characteristic mycelial mode of growth, offers a general method fortheir selective isolation, and does not rely on the use of special nutrient media orof antibacterial antibiotics.

The actinomycetes are a group of bacteriawhich possess many important and interestingfeatures. They are of considerable value asproducers of antibiotics and of other therapeu-tically useful compounds, they exhibit a range oflife cycles which are unique among the procary-otes, and they appear to play a major role in thecycling of organic matter in the soil ecosystem(7, 15).The isolation of actinomycetes from the mixed

microflora present in nature is complicated bytheir characteristic slow growth relative to thatof other soil bacteria. This has resulted in thedevelopment of selective isolation proceduresbased primarily on one or both of the followingapproaches: (i) nutritional selection, in whichmedia are formulated with nutrients which arepreferentially utilized by actinomycetes (4-6, 8-10), and (ii) selective inhibition, in which com-pounds such as antibiotics are incorporated intomedia to selectively inhibit non-actinomycetebacteria (3, 6, 11, 16). The major difficulties witheach of these approaches are that neither isstrictly selective for actinomycetes and thateach has the inherent potential to actually inhibitthe growth of some actinomycetes. We reporthere a method for the isolation of actinomyceteswhich is simple, selective, and based on anentirely new approach.(A preliminary account of this work has been

published [Abstr. Annu. Meet. Am. Soc. Micro-biol. 1982, 112, p. 96].)

MATERIALS AND METHODS

Membrane filters. The membrane filters used wereobtained from Millipore Corp. All filters were 90 mm

in diameter, were composed of mixed esters of cellu-lose, and were steam sterilized before use.Growth conditions. A synthetic agar medium (CMS)

used in most experiments had the following composi-tion: citric acid, 2 mM; MgCI2 * 6H20, 1.25 mM: KC1.10 mM; Na,S04, 2 mM; NaH2PO4 H.O, 10 mM:NH4CI, 100 mM; glucose, 200 mM; 3-(N-morpholino)propanesulfonic acid-tetramethyl ammonium hydrox-ide buffer, pH 7.2. 200 mM; trace elements solution(TS), 10 ml/liter; agar (Difco Laboratories), 20 g; anddistilled water, 1,000 ml. The composition of TS wasas follows: FeClk * 6H.O, 1 mM; MnSO4 * H.O, 2.5mM; CuSO4 - 5H,O, 0.2 mM; CaCl. * 2H20. 10 mM;H,B10, 1 mM; CoCI. * 6H20, 0.1 mM; ZnCI., 0.5 mM:and Na2MoO4 * 2H,O, 0.1 mM. TS was prepared in0.1 N HCl. Glucose, magnesium chloride, and TSwere sterilized and added separately to autoclaved andcooled CMS before the plates were poured. A modifi-cation of Bennett medium was also used in someexperiments and contained, per liter of distilled water,the following: yeast extract, 1.0 g; beef extract, 1.0 g;casein hydrolysate (vitamin and salt free; ICN Phar-maceuticals, Inc.), 2.0 g; 1 M potassium phosphatebuffer, pH 7.0, 30 ml; 50% (wt/vol) glucose, 20 ml; TS.10 ml; and agar (Difco), 20 g. Glucose and TS weresterilized and added separately to the autoclaved andcooled modified Bennett medium before the plateswere poured. All media contained cycloheximide andcandicidin, each at 100 pLg/ml. to suppress the growthof fungi. Cycloheximide stock solution (1.25%[wt/vol]) was prepared in water and sterilized byfiltration. Candicidin stock solution (2.5% [wt/vol])was prepared in N-.N-dimethylformamide. Each anti-biotic was added to sterilized molten agar mediabefore the plates were poured. All incubations werecarried out at 28°C.

Inocula. Samples of soil, water, and various vegeta-ble materials used in experiments were collected local-ly and from different locations around the world.

Electron microscopy. Filters for scanning electronmicroscopy were fixed in 3% glutaraldehyde, rinsed in

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B g DFIG. 1. Membrane filter isolation procedure for actinomycetes. Agar medium was overlaid with a sterile

cellulose ester membrane filter (A), the filter surface was inoculated and the petri plate was incubated untilcolonies developed (B), the filter was removed to expose the actinomycete colonies (arrow) in the underlyingagar (C), and the petri plate was reincubated to allow development of the colonies (D).

phosphate buffer, postfixed in 1%5 osmium tetroxide,and dehydrated with increasing concentrations of eth-anol. The filters were examined by light microscopy,and intact actinomycete colonies were located, ex-cised, and critically point dried. Specimens were af-fixed to aluminium stubs and gold coated, and repre-sentative areas were photographed by scanningelectron microscopy.

Chemicals. Cycloheximide and candicidin were pur-chased from Calbiochem Corp. All other chemicalswere of reagent grade or of the highest purity avail-able.

RESULTSIsolation procedure. Actinomycete colonies

growing on agar media characteristically extendonto and well beneath the agar surface (14). Incontrast, colonies of non-actinomycete bacteria

generally limit their growth to the immediateagar surface. It was expected that selectiveisolation of actinomycetes could be achieved byplacing a suitable barrier between the growingbacterial colonies and the agar surface whichwould allow only actinomycetes to penetrate tothe underlying agar. A cellulose ester membranefilter of proper pore diameter was found toprovide such a barrier. A procedure that usesmembrane filters for the isolation of actinomy-cetes from mixed microbial populations wasdeveloped (Fig. 1). An agar medium was over-laid with a sterile membrane filter (Fig. 1 A)without trapping air between the filter and theagar surface. During application to the agar, thefilter must not be allowed to crack or have itsintegrity otherwise destroyed. Liquid or solid

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ISOLATION OF ACTINOMYCETES 927

TABLE 1. Recovery of actinomycetes bymembrane filter isolation

Pore size % Recovery" at:(uLm) 1 day 2 days 3 days 4 daysh

0.22 0 0 15 1560.30 0 1.8 52 2740.45 0 2.3 97 243

a The percent recovery was calculated as the num-ber of actinomycete colonies present on the agardivided by the number of actinomycete colonies pres-ent on the filter surface, multiplied by 100.

b Recovery of >100% after 4 days reflects thedifficulty of recognizing and counting actinomycetecolonies which had been overgrown by other bacteriaon the filter surface.

inoculum was then applied to the filter surface,and the inoculated plates were incubated untilvisible growth of bacteria occurred (Fig. 1B).The membrane filter was then removed asepti-cally from the agar medium (Fig. 1C), and theplate was reincubated until growth was ob-served. As shown in Fig. 1D, the colonies pre-sent on the plate were exclusively actinomy-cetes. Our experience has shown that twofactors are particularly important in achievingsuccess with the method. (i) Since penetration ofthe membrane filter by actinomycetes is neces-sary to achieve a physical separation of actino-mycete and non-actinomycete colonies, it isimportant to choose culture conditions that willencourage growth and colony formation by thedesired actinomycetes. (ii) The application ofthe inoculum must be limited to the central areaof the filter, maintaining at least a 1.0-cm-widesterile border along the filter edge. This precau-tion will prohibit growth of non-actinomycetebacteria over the edge of the filter and onto theunderlying agar surface.

Effect of filter pore size on actinomycete selec-tion. Membrane filters of different pore sizeswere tested for their ability to select actinomy-cetes exclusively. Plates of CMS and modifiedBennett medium were overlaid with filters of3.0-, 2.0-, 1.0-, 0.65-, 0.45-, 0.30-, 0.22-, 0.10-,0.05-, and 0.01-p.m-pore diameters. The filterswere inoculated with particles of soil and incu-bated at 28°C. After 4 days, the filters wereremoved, and the plates were reincubated for 4days to allow growth of the colonies. The bacte-rial populations selected by the filters of differ-ent pore sizes were evaluated macroscopicallyand microscopically for the presence of non-actinomycete or actinomycete colonies or both.It was found that filters with a pore diameter of>:0.65 p.m did not discriminate between actino-mycetes and other types of bacteria, whereasfilters with a pore diameter of s0.10 p.m blocked

penetration to the agar of all bacteria, even afterincubation for up to 10 days. Only filters in thepore size range of 0.45 to 0.22 p.m were observedto allow exclusive penetration of actinomycetes.

Effect of pore size on the efficiency of recoveryfrom agar of actinomycetes on the filter surface.Replicate plates of CMS overlaid with 0.22-,0.30-, and 0.45-p.m-pore filters were inoculated,and at 1-day intervals, one set of plates wasremoved from incubation and the number ofactinomycete colonies present on each of thethree filters of different pore sizes was counted.The filters were then removed, and the plateswere reincubated to allow development of acti-nomycete colonies on the agar. The number ofcolonies present on the agar was then counted,and the percent recovery was determined asdescribed in footnote a of Table 1. The resultsshow (Table 1) that after 3 days of incubation,the 0.45-p.m-pore filters yielded the highest per-cent recovery of actinomycete colonies. Filtersof 0.30-pm-pore diameter were only about 50%as efficient as 0.45-p.m-pore filters after 3 days,whereas 0.22-p.m-pore filters yielded the lowestpercent recovery.The data in Table 1 also indicate the time

required for the penetration of actinomycetecolonies through the filters. For example, onplates overlaid with 0.45-p.m-pore filters, only a2.3% recovery of actinomycetes was observedafter 2 days of incubation. After 3 days ofincubation, however, almost 100% of the actino-mycetes were recovered on the agar. It appearsthat penetration of the filters by actinomycetesis a process closely related to growth of thecolonies.

Penetration mechanism. The importance of thepore diameter in determining whether a mem-brane filter was penetrated by actinomycetesand the time required for actinomycetes to pene-trate a filter suggested that the penetrationmechanism involved growth of hyphae throughthe filter pores and not digestion of the filtermatrix. This was investigated by scanning elec-tron microscopy of a cross section of an actino-mycete colony which penetrated a 0.22-p.m-poremembrane filter (Fig. 2). It is clear that noobvious change in the filter matrix occurred inthe vicinity of the actinomycete colony, but thecolony penetrated the filter, as evidenced bymycelia on both the top and bottom surfaces ofthe filter. One can conclude that penetration ofthe membrane filter by actinomycetes occurs bygrowth of the branching mycelia through theopenings of the filter matrix.

DISCUSSIONMembrane filters have been used by various

investigators to isolate actinomycetes from wa-

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928 HIRSCH AND CHRISTENSEN

_l~..A BFIG. 2. Scanning electron micrograph of a cross

section of an actinomycete colony which penetrated a

0.22-p.m-pore membrane filter. (T) Top surface of thefilter to which the inoculum was applied; (B) bottomsurface of the filter which was in contact with the agar;(C) actinomycete colony present on the top surface ofthe filter; (D) tufts of mycelia from the colony in (C)which penetrated the filter matrix (F) and protrudefrom the bottom of the filter. Bar, 50 p.m.

ter (1, 2). Trolldenier (12, 13) collected actino-mycete spores from water on a membrane filter,incubated the spore side of the membrane incontact with nutrient agar, and observed theproduction of actinomycete colonies on the re-

verse side of the filter. We describe here a new

procedure that uses membrane filters for theisolation of actinomycetes from mixed microbialpopulations. Actinomycetes are selectively sep-arated from other bacteria by their ability topenetrate the pores of a membrane filter and togrow on an underlying agar medium, whereasthe growth of other bacteria remains restrictedto the filter surface. The ability of actinomycetesto penetrate the tortuous pores of the membranefilter apparently is due to their characteristicgrowth as highly branched mycelial networks.We have isolated many different actinomycetecultures from the environment by using thismethod. Based on colony morphology, culturebehavior, and microscopic observation, it ap-

pears that our isolates include members of theStreptomycetaceae, Micromonosporaceae (me-sophiles and thermophiles), Nocardiaceae, andActinoplanaceae. Thus, this procedure is suit-able for selective isolation of a taxonomic varie-ty of actinomycetes.Among the advantages offered by this tech-

nique are its simplicity and its ability to isolateactinomycetes from a variety of different sam-ples without having to resort to the use of non-discriminating antibacterial antibiotics or re-stricting nutritional conditions. Moreover, thefilter technique can complement existing proce-dures designed for the isolation of actinomy-cetes.

ACKNOWLEDGMENTS

We thank John Nichols and Solomon Scott for help with thescanning electron microscopy.

LITERATURE CITED

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7. Lacey, J. 1978. Ecology of actinomycetes in fodder andrelated subtrates, p. 161-168. In M. Mordarski, W. Kur-ylowicz, and J. Jeljaszewicz (ed.). Nocardia and Strepto-myces: proceedings of the international symposium onNocardia and Streptomyces, Warsaw, October 1976. Gus-tav Fischer Verlag, Stuttgart.

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