New method for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22-0.45µm pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of non actinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was re incubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.

Keyword: actinomycetes, cellulose ester membrane filter, isolation, penetration mechanism

The actinomycetes are a group of bacteria which possess many important and interesting features. They are of considerable value as producers of antibiotics and of other therapeutically useful compounds, they exhibit a range of life cycles which are unique among the prokaryotes, and they appear to play a major role in the cycling of organic matter in the soil ecosystem.^1‒3 The isolation of actinomycetes from the mixed microflora present in nature is complicated by their characteristic slow growth relative to that of other soil bacteria. This has resulted in the development of selective isolation procedures based primarily on one or both of the following approaches: (i) nutritional selection, in which media are formulated with nutrients which are preferentially utilized by actinomycetes.^4,5 ii) selective inhibition, in which com-pounds such as antibiotics are incorporated into media to selectively inhibit non-actinomycete bacteria.^6‒8 The major difficulties with each of these approaches are that neither is strictly selective for actinomycetes and that each has the inherent potential to actually inhibit the growth of some actinomycetes.⁴ Present study reports a method for the isolation of actinomycetes which is simple, selective, and based on an entirely new approach.

The membrane filters used were obtained from Millipore Corp. All filters were 90mm in diameter, were composed of mixed esters of cellulose, and were steam sterilized before use.

A synthetic agar medium (SAM) used in most experiments had the following composition: citric acid, 2mM; MgCl₂.6H₂O, 1.25 mM; KCl, 10 mM: Na₂SO₄, 2 mM; NaHPO₄.H₂O, 10 mM; NH₄CI, 100 mM; glucose, 200 mM: 3-(N-morpholino) propanesulfonic acid-tetramethyl ammonium hydroxide buffer, pH 7.2, 200 mM: trace elements solution (TS), 10 ml/liter; agar, 20 g: and distilled water, 1,000 ml. The composition of TS was as follows: FeCI₃.6H₂O, 1 mM: MnSO₄.H₂O, 2.5 mM; CuSO₄.5H₂0, 0.2 mM: CaCl₂.2H₂0, 10 mM: H₃B0₃, 1 mM; CoCl₂.6H₂O, 0.1 mM: ZnCI₂, 0.5 mM: and Na₂Mo0₄.2H₂0, 0.1 mM. TS was prepared in 0.1 N HCI. Glucose, magnesium chloride, and TS were sterilized and added separately to autoclaved and cooled SAM before the plates were poured. A modification of Starch casein agar medium was also used in some experiments and contained, per liter of distilled water the following ingredients yeast extract, 1.0 g: beef extract, 1.0 g: casein hydrolysate, 2.0g; 1 M potassium phosphate buffer. pH 7.0. 30 ml: 50% (wt/vol) glucose, 20 ml: TS,10 ml; and agar, 20g. Glucose and TS were sterilized and added separately to the autoclaved and cooled modified Bennett medium before the plates were poured. All media contained cycloheximide at 100µg/ml to suppress the growth of fungi. Cycloheximide stock solution (1.25%[wt/vol]) was prepared in water and sterilized by filtration.⁹ An antibiotic was added to sterilized molten agar media before the plates were poured. All incubations were carried out at 28°C. Soil samples were drawn from tobacco growing soils. Chemicals used were of reagent grade or of the highest purity available.

Isolation procedure

Actinomycete colonies growing on agar media characteristically extend onto and well beneath the agar surface. In contrast, colonies of non-actinomycete bacteria generally limit their growth to the immediate agar surface. It was expected that selective isolation of actinomycetes could be achieved by placing a suitable barrier between the growing bacterial colonies and the agar surface which would allow only actinomycetes to penetrate to the underlying agar. A cellulose ester membrane filter of proper pore diameter was found to provide such a barrier. A procedure that uses membrane filters for the isolation of actinomycetes from mixed microbial populations was developed (Figure 1). An agar medium was over-laid with a sterile membrane filter (Figure 1A) without trapping air between the filter and the agar surface. During application to the agar, the filter must not be allowed to crack or have its integrity otherwise destroyed. Liquid or solid inoculum was then applied to the filter surface, and the inoculated plates were incubated until visible growth of bacteria occurred (Figure 1B). The membrane filter was then removed aseptically from the agar medium (Figure 1C), and the plate was reincubated until growth was ob-served. As shown in Figure 1D, the colonies present on the plate were exclusively actinomycetes. Present studies revealed that two factors are particularly important in achieving success with the method.

1. Since penetration of the membrane filter by actinomycetes is necessary to achieve a physical separation of actinomycete and non-actinomycete colonies, it is important to choose culture conditions that will encourage growth and colony formation by the desired actinomycetes.
2. The application of the inoculum must be limited to the central area of the filter, maintaining at least a 1.0-cm-wide sterile border along the filter edge. This precaution will prohibit growth of non-actinomycete bacteria over the edge of the filter and onto the underlying agar surface.

Figure 1 Membrane filter isolation procedure for actinomycetes. (A) Agar medium was overlaid with a sterile cellulose ester membrane filter. (B) The filter surface was inoculated and the petriplate was incubated until colonies developed. (C) The filter was removed to expose the actinomycetes colonies (arrow) in the underlying agar. (D) The petriplate was reincubated to allow development of the colonies.

Effect of filter pore size on actinomycete selection

Membrane filters of different pore sizes were tested for their ability to select actinomycetes exclusively. Plates of SAM and modified Bennett medium were overlaid with filters of 3.0-, 2.0-, 1.0-, 0.65-, 0.45-, 0.30-, 0.22-, 0.10-, and 0.05-, p.m-pore diameters. The filters were inoculated with particles of soil and incubated at 28°C. After 4 days, the filters were removed, and the plates were re incubated for 4 days to allow growth of the colonies. The bacterial populations selected by the filters of different pore sizes were evaluated macroscopically and microscopically for the presence of non-actinomycete or actinomycete colonies or both. It was found that filters with a pore diameter of <0.10 p.m did not discriminate between actinomycetes and other types of bacteria, whereas filters with a pore diameter of <0.10 p.m blocked penetration to the agar of all bacteria, even after incubation for up to 12 days. Only filters in the pore size range of 0.45 to 0.22p.m were observed to allow exclusive penetration of actinomycetes.

Effect of pore size on the efficiency of recovery from agar of actinomycetes on the filter surface

Replicate plates of SAM overlaid with 0.22-, 0.30- and 0.45-p.m-pore filters were inoculated, and at 1-day intervals, one set of plate was removed from incubation and the number of actinomycete colonies present on each of the three filters of different pore sizes was counted. The filters were then removed, and the plates were reincubated to allow development of actinomycete colonies on the agar. The number of colonies present on the agar was then counted, and the percent recovery was determined as described in footnote of Table 1. The results show Table 1 that after 3 days of incubation, the 0.45-p.m-pore filters yielded the highest per-cent recovery of actinomycete colonies. Filters of 0.30-µm-pore diameter were only about 50% as efficient as 0.45-µm-pore filters after 3 days, whereas 0.22-p.m-pore filters yielded the lowest percent recovery. 

Penetration mechanism

The importance of the pore diameter in determining whether a membrane filter was penetrated by actinomycetes and the time required for actinomycetes to penetrate a filter suggested that the penetration mechanism involved growth of hyphae through the filter pores and not digestion of the filter matrix. It is clear that no obvious change in the filter matrix occurred in the vicinity of the actinomycete colony, but the colony penetrated the filter, as evidenced by mycelia on both the top and bottom surfaces of the filter. One can conclude that penetration of the membrane filter by actinomycetes occurs by growth of the branching mycelia through the openings of the filter matrix. Membrane filters have been used by various investigators to isolate actinomycetes from water.¹⁰ Trolldenier¹¹ collected actinomycete spores from water on a membrane filter, incubated the spore side of the membrane in contact with nutrient agar, and observed the production of actinomycete colonies on the reverse side of the filter. Present study reports a new procedure that uses membrane filters for the isolation of actinomycetes from mixed microbial populations. Actinomycetes are selectively separated from other bacteria by their ability to penetrate the pores of a membrane filter and to grow on an underlying agar medium, whereas the growth of other bacteria remains restricted to the filter surface. The ability of actinomycetes to penetrate the tortuous pores of the membrane filter apparently is due to their characteristic growth as highly branched mycelial networks. Based on colony morphology, culture behavior, and microscopic observation, it appears that our isolates include members of the Streptomycetaceae, Micromonosporaceae, Nocardiaceae, and Actinoplanaceae. Thus, this procedure is suitable for selective isolation of a taxonomic variety of actinomycetes. Among the advantages offered by this technique are its simplicity and its ability to isolate actinomycetes from a variety of different samples without having to resort to the use of non-discriminating antibacterial antibiotics or restricting nutritional conditions. Moreover, the filter technique can complement existing procedures designed for the isolation of actinomycetes (Table 1).

*The percent recovery was calculated as the number of actinomycete colonies present on the agar divided by the number of actinomycete colonies present on the filter surface, multiplied by 100.

∞Recovery of >100% after 4 days reflects the difficulty of recognizing and counting actinomycete colonies which had been overgrown by other bacteria on the filter surface (Figure 1).

None.

The authors declared there is no conflict of interest.

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